Efficacy of a Spearmint (Mentha spicata L.) Extract as Nutritional Support in a Rat Model of Hypertensive Glaucoma

Abstract

Purpose: Glaucoma is an eye–brain axis disorder characterized by loss of retinal ganglion cells (RGCs). Although the role of intraocular pressure (IOP) elevation in glaucoma has been established, the reduction of oxidative stress and inflammation has emerged as a promising target for neuronal tissue–supporting glaucoma management. Therefore, we evaluated the effect of a proprietary spearmint extract (SPE) on RGC density, activity, and neuronal health markers in a rat model of hypertensive glaucoma.

Methods: Animals were divided in four groups: untreated healthy control and three glaucomatous groups receiving orally administered vehicle, SPE-low dose, or SPE-high dose for 28 days. Ocular hypertension was induced through intracameral injection of methylcellulose at day 15. At day 29, rats underwent electroretinogram (ERG) recordings, and retinas were analyzed for RGC density and markers of neural trophism, oxidative stress, and inflammation.

Results: SPE exerted dose-dependent response benefits on all markers except for IOP elevation. SPE significantly improved RGC-related ERG responses, cell density, neurotrophins, oxidative stress, and inflammation markers. Also, in SPE-high rats, most of the parameters were not statistically different from those of healthy controls.

Conclusions: SPE, a plant-based, polyphenolic extract, could be an effective nutritional support for neuronal tissues.

Translational Relevance: These results suggest that SPE not only may be a complementary approach in support to hypotensive treatments for the management of glaucoma but may also serve as nutritional support in other ocular conditions where antioxidant, anti-inflammatory, and neuroprotective mechanism are often disrupted.

Introduction

Glaucoma, a neurodegenerative disorder, is one of the leading causes of blindness and is estimated to affect more than 76 million people worldwide, with that number expected to reach over 111 million by 2040.¹ The disease affects both the eye and brain with typically irreversible neural damage. In its various subtypes (primary open-angle glaucoma, primary angle-closure glaucoma, normotensive glaucoma, etc.), its global prevalence is estimated at 3.5% of the population 40 to 80 years of age.² Data from a meta-analysis conducted in 2022 reported a pooled prevalence estimate of primary open-angle glaucoma in Europe of 2.60% (95% confidence interval, 1.90–3.56) with expected growth associated with a progressively aging population in Western countries.³ Glaucoma is mainly characterized by a progressive optic neuropathy, including chronic axonal damage and retinal ganglion cell (RGC) loss. The complex pathophysiology behind the RGC degeneration is comprised of a series of genetic, metabolic, and environmental factors and consequently requires a multifactorial approach to glaucoma management.⁴ 

Intraocular pressure (IOP) elevation represents one of the most recurrent risk factors driving glaucomatous progression and RGC neurodegeneration.⁵ Thus, the reduction of ocular hypertension represents the main target for the pharmaceutical treatments currently in use with significant but not resolutive effects.⁶ This highlights the significance of contributing pathophysiological mechanisms other than IOP elevation that equally drive the disease progression or etiology (such as in normotensive glaucoma). This necessitates the understanding of these other mechanisms and the development of solutions to mitigate these effects. 

Current research advancing this understanding links glaucoma to degeneration of the central nervous system (CNS), moving it away from being considered just a traditional eye disease. A recent review assessing the morphological and functional changes affecting the CNS in glaucoma⁷ reported structural alterations along the visual pathway and in numerous cognitive functions outside the visual pathway, such as for visual memory, working memory, attention, and motor coordination. These observations suggest that independent mechanisms of neurodegeneration in glaucoma are potentially affecting neural tissues in addition to the known anterograde and/or retrograde neuronal degeneration.^7–9 Recent evidence also suggests that metabolic stress and bioenergetic insufficiency play key roles in the progression of glaucoma ultimately resulting in RGC degeneration through the promotion of oxidative stress and inflammation.^10,11 Furthermore, levels of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) could play a key role in the progression of glaucoma.¹² BDNF indeed plays a vital role in RGC physiology, protecting RGCs in conditions of ocular hypertension, hypoxia, or glucose deprivation, whereas the role of NGF is less clear, as NGF binds two different receptors that evoke opposite effects.¹³ Nonetheless, when neurotrophin support is lacking, the induction of apoptotic death of RGCs has been described.¹⁴ RGCs are supplied with neurotrophins produced locally in the retina but also in the brain, and these neurotrophins are delivered to RGCs through retrograde transport, which is impaired in the early stages of glaucoma, thus leading to a deficiency in the trophic support of RGCs.¹² Therefore, the use of antioxidant, anti-inflammatory, and/or neurotrophic support solutions as complementary approaches for the management of this condition is gaining increasing attention. In this context, a proprietary spearmint extract (SPE), marketed as Neumentix (Kemin Foods, L.C., Des Moines, IA), which is a polyphenol-rich ingredient derived from patented lines of spearmint (Mentha spicata L.) plants,^15–17 could provide suitable nutritional support. Multiple preclinical studies with SPE have demonstrated that SPE can reduce markers of oxidative stress and inflammation and promote neuroprotection in neural tissue.^18–20 Furthermore, SPE has been shown to modulate brain neurotransmitters in the mouse and promote neurogenesis in rat hippocampal neurons.^21,22 Clinical research has additionally shown that the extract is well tolerated and supports cognitive function improvements in both healthy young and older people.^23–27 Specifically, the oral administration of SPE for 12 weeks has been shown to improve working memory, attention, and agility compared to placebo. Additionally, SPE in combination with green tea extract has been shown to improve sleep quality and next-day concentration, decision-making, working memory, and reaction time in healthy young people. Furthermore, there is biological plausibility that SPE offers cognitive and sleep benefits by supporting the underlying health of neuronal tissue. Recent animal models of glaucomatous conditions induced by optic nerve crush or methylcellulose (MCE)-induced ocular hypertension indicate that dietary interventions including SPE counteract the inflammatory processes and morpho-functional alterations of glaucoma.^28,29 These studies suggest that SPE could be a potential candidate for providing nutritional support for ocular neural tissues. 

Hence, the objective of this study was to determine the effect of oral supplementation of SPE at two doses on RGC degeneration in terms of retinal dysfunction and cell loss, following IOP elevation in a rat model of MCE-induced hypertensive glaucoma. In this model, the increase in IOP results in a sudden elevation of inflammatory and oxidative stress markers that in glaucoma patients may require a considerable amount of time to occur. Nevertheless, it can still provide valuable information about the mechanisms involved in glaucoma and preliminary data on how they can potentially be modulated by nutritional intervention. Scotopic and photopic electroretinogram (ERG) and pattern ERG (PERG) were used to evaluate retinal function, and RGC density was measured by immunohistochemistry for RNA-binding protein with multiple splicing (RBPMS), a selective RGC marker in the mammalian retina.³⁰ Levels of neurotrophins, BDNF, and NGF, which are known to promote neuronal survival and synapsis plasticity in the CNS^31,32 and RGC survival,¹³ were measured to assess direct neuroprotective effects. Finally, levels of markers of oxidative stress, of cellular antioxidant defense, and of neuroinflammation in the retina were measured to ascertain the antioxidant and anti-inflammatory effects of the SPE intervention. 

Materials and Methods

Animals

Forty male Sprague Dawley rats (8 weeks old, about 300 g) were purchased from Envigo Italy (San Pietro al Natisone, Italy). Each rat was kept in a regulated environment (23°C ± 1°C, 50% ± 5% humidity) with 12-hour light/dark cycles (lights on at 08:00 AM) and fed with a standard diet (2018 Teklad Global Diets; Envigo Italy) and water ad libitum. Rats were housed one per cage to avoid motor restraints. Before the study started, all rats were acclimatized for 7 days to handling and tonometry. Animals were managed in agreement with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The study follows the European Communities Council Directive (2010/63/UE) and the Italian guidelines for animal care (DL 26/14). The experimental protocol was authorized by the Commission for Animal Wellbeing of the University of Pisa (protocol no. 4/2022) and by the Ministry of Health (protocol no. 307/2022-PR). The principles of the 3Rs (replacement, reduction, and refinement) for ethical use of animals in scientific research were utilized to reduce both the number and suffering of the animals. 

Rats were randomly divided into four groups as follows: one group (10 rats) of healthy controls (control, rats receiving no MCE injection and no supplementation; group 1), and three groups of glaucomatous rats (10 rats/group) receiving MCE injection and randomized to oral supplementation with vehicle (MCE + vehicle; group 2); low-dose SPE (MCE + SPE-low; group 3); and high-dose SPE (MCE + SPE-high; group 4). 

Rat Model of Hypertensive Glaucoma

The induction of ocular hypertension was performed in agreement with published procedures.^33,34 Briefly, 2% MCE w/v in sterile saline was prepared to obtain a solution viscosity ranging from 3500 to 5600 cps. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg/kg) and injected into the anterior chamber with 15 µL of the MCE solution in both eyes using a Hamilton syringe equipped with an 18-gauge needle. The needle was inserted in the iridocorneal angle at about 1 mm from the ora serrata and oriented parallel to the iris surface. After the slow injection of MCE (1-minute duration), the needle was kept in place for 1 minute to avoid MCE outpours. Immediately after the injection of MCE, antibiotic eyedrops were instilled in order to prevent the occurrence of endophthalmitis. The animals were kept under continuous monitoring during the whole period of anesthesia for possible alterations in the breathing frequency up to the complete recovery. Each rat was daily monitored during the following 3 days for any alterations in ocular tissues (corneal opacity, cataracts, conjunctivitis, or hyphema). 

Dietary Supplementation

SPE (marketed as Neumentix by Kemin Foods, L.C., Des Moines, IA) is a proprietary ingredient sourced from patented, non–genetically modified organism (GMO) lines of native spearmint (Mentha spicata L.) grown in the United States and developed by using traditional plant breeding methodologies. These plants are capable of accumulating >100 mg/g rosmarinic acid (RA) on a dry-weight basis, a much higher concentration compared to that reported for traditional spearmint, with RA content in the range of 7.1 to 58.5 mg/g.^15,35 Ultra-high performance liquid chromatography–mass spectrometry analysis of SPE shows that the product has a characteristic and consistent phenolic fingerprint and contains a powerful assortment of phenolic compounds including, but not limited to, RA, salvianolic acid, caffeic acid, caftaric acid, quinic acid, and lithospermic acid.¹⁵ SPE is standardized to 14.5% to 17.5% RA and 24% to 37% total phenolics. 

In the present study, SPE was tested at low (SPE-low) and high (SPE-high) doses corresponding to half or full dosages found to exert cognitive benefits in clinical trials,^23–25,27 taking into account the difference in the metabolism of the two species (man and rat)³⁶ and the dose used in previous preclinical studies.^18–21 SPE-high was administered at 93.0 mg/kg body weight per day, corresponding to a human daily dose of 15 mg/kg body weight (900 mg/d for a 60-kg person). SPE-low was administered at 46.5 mg/kg body weight per day, corresponding to a human daily dose of 7.5 mg/kg body weight (450 mg/d for a 60-kg person). High- and low-dose solutions of SPE were freshly prepared every day in distilled water immediately before the treatment. In particular, SPE was dissolved at 93 mg/mL in distilled water (vehicle) in order to obtain the stock solution to be administered for the high dose. Hence, the low-dose solution containing SPE at 46.5 mg/mL was prepared by performing 1:2 dilution of the high-dose solution. Equal amounts of low- and high-dose solutions (volume administered 300 µL) were administered daily by oral gavage for 14 days before and 14 days after the MCE injection. Animals were routinely observed during the treatment period both before and after the MCE injection. In particular, eating and drinking behaviors and the possible occurrence of postural signs of distress (hunching, huddling, crouching, rigidity) were monitored up to 15 minutes after administration of the product. 

Measurement of IOP

A time-dependent IOP profile was built for each group in order to test the effect of the oral supplementation on the ocular hypertension. IOP was non-invasively assessed daily using rebound tonometry (Tonolab; iCare Finland Oy, Helsinki, Finland) before and after MCE injections in every group for the period under investigation (28 days). Multiple sampling procedures (5–10 readings) were performed at the same range of time during the day. 

Measurement of RGC Activity

The effect of the oral supplementation with SPE on the glaucomatous RGC dysfunction was analyzed at day 29 with ERG recording, in agreement with published procedures.³⁷ In particular, two main functional output related with RGC activity were analyzed: photopic negative response (PhNR) and PERG. These two procedures provide information regarding the inner retinal response in the context of the overall light-adapted retinal activity (PhNR) and specific RGC activity (PERG). These two parameters were analyzed both per se and in relation with the overall retinal activity as assessed by scotopic ERG. The ERG recording was performed at the endpoint of the study using a commercially available setup (Retimax Advanced; CSO, Firenze, Italy). After dark adaptation overnight, each rat was anesthetized by an intraperitoneal injection of sodium pentobarbital (30 mg/kg) and gently restrained in a custom-made holder with an unobstructed visual field. ERG responses were recorded using silver/silver chloride corneal electrodes. A reference electrode was inserted on the forehead, and a ground electrode was inserted at the base of the tail. Corneal moisture was maintained along the ERG routine by instilling balanced salt solution every 15 minutes. Scotopic ERG responses were retrieved following a single 10-cd·s/m² flash stimulus over a dark background delivered by a Ganzfeld light source. Then, the rats underwent light adaptation to 30-cd·s/m² rod-saturating background light for 10 minutes before photopic ERG responses were recorded using a 3-cd·s/m² stimulus delivered over the same background light. The responses to 10 consecutive stimuli with an interstimulus interval of 3 seconds were recorded and averaged. PERG recordings were then performed by delivering pattern stimuli consisting of 0.05-c/° black and white bars reversing at 1 Hz presented at 98% contrast. The pattern stimuli were administered through a light-emitting diode display with a mean luminance of 50 cd/m² aligned at about 20 cm from the corneal surface. A total of 200 signals were averaged. The ERG waveforms were analyzed for their consistency and evaluated for signal processing and noise filtering. In the photopic ERG waveforms, the PhNR was identified as the first negative deflection after the b-wave. The PERG waveforms were analyzed using Retimax Scientific 7.0.4 software (CSO) to retrieve the amplitude of positive (N35–P50) and negative (P50–N95) components. The amplitudes of PhNR and PERG and the latency of PERG were considered to be analytical parameters related to RGC function. 

Retina Collection

Immediately after the ERG recordings, the rats were euthanized, and the retinas were dissected from other ocular tissues by microsurgical procedures. One retina from each mouse was used for immunohistochemistry; the other was divided in four quadrants that were immediately frozen in liquid nitrogen. For each retina, the four quadrants were randomly assigned to one of four groups: group 1, for western blot and the evaluation of 4-hydroxynonenal (4-HNE) levels; group 2, for the evaluation of malondialdehyde (MDA) levels; group 3, for the evaluation of 8-hydroxy-deoxyguanosine (8-OH-dG) levels; group 4, for the evaluation of glutathione (GSH) levels. 

RGC Immunohistochemistry and Quantification

The effect of SPE on the glaucomatous RGC loss was evaluated by analyzing the RGC density using immunofluorescence. Briefly, isolated retinas were immersion-fixed in 4% w/v paraformaldehyde and stored at 4°C. Retinas underwent whole-mount immunostaining for RBPMS, using a guinea pig polyclonal antibody (ABN1376; Merck KGaA, Darmstadt, Germany) at 1:100 dilution for 72 hours, followed by 48-hour incubation with a fluorescein-conjugated donkey anti-guinea pig secondary antibody (AP193F; Merck KGaA) at 1:80 dilution. After being processed, retinas were analyzed with an epifluorescence microscope (Ni-E; Nikon Europe, Amsterdam, The Netherlands) equipped with a 20 × plan achromat objective, a digital camera (DS-Fi1c; Nikon Europe), and a motorized stage for organ whole-mount reconstruction. The derived images were automatically analyzed for the RBPMS-positive cell density following the sampling of four radially opposite images at two different radial eccentricities (center = 0.5 mm, periphery = 4 mm from the optic disc) in order to determine the average density of RGCs in peripheral and central retina. 

Western Blot

Retina quadrants were lysed with radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology, Dallas, TX) added with phosphatase and proteinase inhibitor cocktails (1:100 dilution; Roche Applied Science, Indianapolis, IN), and protein concentration was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). For each sample, 20 µg of proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 4% to 20% polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA) that were subsequently transblotted onto nitrocellulose membranes (Bio-Rad Laboratories). Blots were blocked with 5% skim milk for 1 hour and then individually incubated overnight with the following primary antibodies: rabbit monoclonal raised against BDNF (1:1000 dilution, ab108319; Abcam, Cambridge, UK) or NGF (1:1000 dilution, ab52918; Abcam); rabbit polyclonal raised against nuclear factor erythroid 2–related factor 2 (Nrf2; 1:300 dilution, ab92946; Abcam), heme oxygenase-1 (HO-1; 1:500 dilution, ab13243; Abcam), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; 1:1000 dilution, ab16502; Abcam), or the phosphorylated form of NF-κB (pNF-κB; 1:1000 dilution, sc-33020; Santa Cruz Biotechnology); mouse monoclonal raised against interleukin (IL)-6 (1:500 dilution; sc-57315; Santa Cruz Biotechnology) or β-actin (1:2500 dilution, A2228; Merck KGaA); hamster monoclonal raised against IL-1β (1:100 dilution, sc-12742; Santa Cruz Biotechnology); or goat polyclonal antibody raised against IL-10 (1:100 dilution, sc-1783; Santa Cruz Biotechnology). Blots were then incubated for 2 hours with the appropriate horseradish peroxidase–conjugated secondary anti-rabbit, anti-mouse, anti-hamster, or anti-goat antibodies, diluted 1:5000 (Santa Cruz Biotechnology). Membranes were visualized using the Clarity Western ECL Substrate (Bio-Rad Laboratories). Images were acquired using the ChemiDoc XRS+ (Bio-Rad Laboratories). The optical density (OD) relative to the target bands was evaluated (Image Lab 3.0 software; Bio-Rad Laboratories) and normalized to the corresponding OD of β-actin or NF-κB as appropriate. 

Measurement of Oxidative Stress Markers

Oxidative stress in the retina was measured by evaluating the levels of common markers. MDA and 4-HNE are products of lipid peroxidation, and 8-OH-dG is generated by oxidative damage to DNA; their levels have been found to increase in glaucoma patients.³⁸ GSH is a major endogenous antioxidant in the retina whose levels have been found to decrease in both hypertensive and normotensive glaucoma patients.³⁹ The levels of MDA, 4-HNE, 8-OH-dG, and GSH were evaluated using commercially available kits: Lipid Peroxidation (MDA) Assay Kit (ab118970, Abcam), Lipid Peroxidation (4-HNE) Assay Kit (ab238538, Abcam), 8-Hydroxy 2 Deoxyguanosine ELISA Kit (ab201734, Abcam), and GSH+GSSG/GSH Assay Kit (Colorimetric) (ab239709, Abcam), according to the manufacturer's instructions. 

Data Analysis

In adherence with the 3R principles for the ethical use of animals in scientific research, a priori power analysis was performed (G*Power 3.0.10, www.gpower.hhu.de). Sample size was calculated considering α = 0.05, an effect size of at least 0.6 (an effect size sufficiently high to evaluate relevant differences among groups), and a statistical power of at least 0.80. Animal treatments were performed in a non-blinded fashion. Investigators performing ERG, immunohistochemistry, western blot, and measurement of oxidative stress markers were blinded to the treatment group. Blinding was performed by assigning a numerical coded identifier to the samples. Unblinding was done after data collection. Data analysis of IOP and ERG results provided the average of the two eyes as representative measures from each rat. Data were analyzed by the Shapiro–Wilk test to verify their normal distribution. Statistical significance was evaluated with Prism 8.0.2 (GraphPad Software, San Diego, CA) using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Data are expressed as means ± SEM of the reported n values. Differences with P < 0.05 were considered statistically significant. 

Results

Spearmint Extract Does Not Affect MCE-Induced Ocular Hypertension

Animals were routinely observed during the treatment period both before and after the MCE injection. In particular, eating and drinking behaviors and the possible occurrence of postural signs of distress were monitored up to 15 minutes after administration of the product. As shown in Figure 1, the IOP profiles in all of the MCE-treated rats, assessed by rebound tonometry, revealed a significant increment in IOP levels, reaching a peak of about 34 mmHg within 24 hours after the MCE injection. Hereinafter, IOP gradually decreased over time, although it maintained higher levels as compared to healthy controls, thus confirming the reliability of the model in reproducing a glaucomatous-like ocular hypertension throughout the time window under analysis. Glaucomatous rats treated with either low- or high-dose SPE displayed IOP profiles comparable to those of vehicle-treated glaucomatous controls, without any significant difference in either the early IOP peak or its following gradual decrement. 

Figure 1.

 

Values of intraocular pressure. MCE injection increased IOP and SPE did not affect increased IOP. Data are shown as mean ± SEM (n = 10 for each group). Day 0 corresponds to the day of MCE intraocular injection. Healthy controls did not receive a MCE injection.

Spearmint Extract Exerts a Dose-Dependent Beneficial Effect in RGC-Related ERG Parameters

Scotopic ERG was performed to assess the overall photoreceptor activity, reflected by the scotopic a-wave, and the overall post-receptor activity, reflected by the scotopic b-wave, at the end of the study. As expected, neither scotopic ERG parameter was significantly affected following either the MCE injection or the supplementation regimens (Figs. 2A–2C). Similarly, the photopic b-wave, reflecting the cone-specific overall post-receptor activity, displayed no alterations in amplitude in any experimental group (Figs. 3A, 3B). In contrast, the PhNR amplitude, reflecting RGC-specific activity, was significantly decreased in vehicle-treated MCE rats as compared to the healthy controls (Figs. 3A, 3C). The loss in PhNR amplitude was dose-dependently attenuated following SPE intervention. The definitive establishment of RGC functional alterations in glaucomatous rats derives from assessment of PERG responses. In vehicle-treated MCE rats, amplitudes in both N35–P50 and P50–N95 components were halved compared to the healthy controls (Fig. 4). SPE administration resulted in significant dose-dependent protection of the amplitude of both N35–P50 and P50–N95 components of the PERG response. 

Figure 2.

 

SPE did not affect scotopic ERG. (A) Representative ERG waveforms recorded at a light intensity of 10 cd·s/m² in control rats and in rats injected with MCE fed with either vehicle or SPE. (B, C) Scotopic a-wave (B) and b-wave (C) amplitudes. Photoreceptor and post-receptor responses to a light flash were not influenced by MCE or SPE, as evidenced by the invariance of both a- and b-wave amplitudes. Data are shown as box plots with minimum to maximum whiskers (n = 10 for each group).

Figure 3.

 

SPE did not affect photopic b-wave amplitude, but it attenuated the MCE-induced reduction of PhNR amplitude. (A) Representative ERG waveforms recorded using a 3 cd·s/m² stimulus on a 30 cd·s/m² rod-saturating background light in control rats and in rats injected with MCE which were fed with either vehicle or SPE. (B) Photopic b-wave amplitude. (C) PhNR amplitude. MCE reduced PhNR amplitude, an effect that was attenuated by SPE in a dose-dependent manner. Data are shown as box plots with minimum to maximum whiskers (n = 10 for each group). ^**P < 0.01and ^****P < 0.0001 versus control; ^§§§P < 0.001and ^§§§§P < 0.0001 versus MCE; ^###P < 0.001 versus SPE-low (one-way ANOVA followed by the multiple-comparison Tukey's test).

Figure 4.

 

SPE attenuated the MCE-induced reduction of PERG amplitude. (A) Representative PERG traces showing the two negative peaks (N35 and N95) and the positive peak P50 in control rats and in rats injected with MCE fed with either vehicle or SPE. (B, C) Mean amplitudes of the N35–P50 (B) and P50–N95 (C) waves. MCE reduced the amplitude of both waves, an effect that was substantially attenuated by SPE in a dose dependent manner. Data are shown as box plots with minimum to maximum whiskers (n = 10 for each group). ^***P < 0.001 and ^****P < 0.0001 versus control; ^§§§§P < 0.0001 versus MCE; ^#P < 0.05 and ^###P < 0.001 versus SPE-low (one-way ANOVA followed by the multiple-comparison Tukey's test).

Spearmint Extract Preserves RGC Density Following MCE-Induced IOP Elevation in a Dose-Dependent Manner

As shown in Figure 5, the immunostaining of RBPMS in whole-mount retinas revealed the typical difference in RGC density between the peripheral and central portion of the retina. The intracameral injection of MCE produced a proportional loss of RGC density in both central and peripheral retina. Oral supplementation with SPE resulted in a significant dose-dependent preservation of RGC density at both central and peripheral retinal locations. Furthermore, in glaucomatous rats supplemented with high-dose SPE, RGC density in the central retina was not statistically different from that in healthy controls. 

Figure 5.

 

SPE prevented the MCE-induced reduction of RGC density. (A) Representative images of RBPMS staining in central and peripheral areas of retinas from control rats and rats injected with MCE which were fed with either vehicle or SPE. Scale bar: 100 µm. (B) Analysis of RBPMS-positive cell density, differentially sampled from the peripheral and central areas of the retina. MCE reduced RGC density, an effect that was attenuated or prevented by SPE in a dose-dependent manner. Data are shown as box plots with minimum to maximum whiskers (n = 10 for each group). ***P < 0.001 and ****P < 0.0001 versus control; ^§§P < 0.01 and ^§§§§P < 0.0001 versus MCE; ^###P < 0.0001 versus SPE dose (one-way ANOVA followed by the multiple-comparison Tukey's test).

Spearmint Extract Promotes the Maintenance of Neural Trophism Under MCE-Induced Glaucomatous Stress in a Dose-Dependent Manner

Neural trophism was evaluated by analyzing the protein levels of BDNF and NGF as crucial neurotrophins involved in the maintenance of RGC viability. As demonstrated by the western blot analysis shown in Figure 6, retinal levels of both BDNF and NGF significantly decreased after the intracameral injection of MCE compared to levels observed in healthy control rats. As compared to vehicle-treated MCE rats, animals receiving SPE at both doses displayed a significant dose-dependent increase in neurotrophin supply. 

Figure 6.

 

SPE attenuated the MCE-induced reduction in neurotrophin levels. (A) Representative western blots from retinal homogenates of control rats or rats that received MCE which were fed with either vehicle or SPE. (B, C) Densitometric analysis of the levels of BDNF (B) and NGF (C). MCE resulted in decreased levels of both BDNF and NGF, effects that were substantially attenuated by SPE in a dose-dependent manner. Data are shown as box plots with minimum to maximum whiskers (n = 10 for each group). ^***P < 0.001 and ^****P < 0.0001 versus control; ^§§P < 0.01, ^§§§P < 0.001, and ^§§§§P < 0.0001 versus MCE; ^##P < 0.01 and ^####P < 0.0001 versus SPE dose (one-way ANOVA followed by the multiple-comparison Tukey's test).

Spearmint Extract Counteracts the MCE-Induced Oxidative Stress in a Dose-Dependent Manner

The effect of SPE in counteracting MCE-induced oxidative stress was analyzed by colorimetric analyses and western blot. In particular, the colorimetric analysis was performed to evaluate the levels of oxidation-deriving products including MDA, 8-OH-dG, and 4-HNE, as well as depletion of the endogenous antioxidant GSH. Western blot was used to assess the levels of Nrf2, the master transcriptional regulator of genes involved in cellular antioxidant response, and of HO-1, one of the main Nrf2 target genes (Fig. 7). Retinal levels of MDA (Fig. 7A), 8-OH-dG (Fig. 7B), and 4-HNE (Fig. 7C) were significantly increased in rats receiving the intracameral injection of MCE, paralleled by a significant depletion of GSH (Fig. 7D). SPE resulted in a statistically significant dose-dependent inhibition of oxidative stress-related phenomena, with the high SPE dose able to restore 4-HNE and GSH to healthy control levels. The MCE-induced increase in oxidative products was reflected in the induction of the cellular antioxidant response as demonstrated by the rise in Nrf2 (Figs. 7E, 7F) and subsequent increase in HO-1 (Figs. 7E, 7G) protein levels. The effect of SPE in counteracting MCE-induced oxidative stress status was also supported by the related dose-dependent attenuation of the cellular antioxidant response. MCE-induced increments of HO-1 levels were substantially attenuated by low- and high-dose SPE. Furthermore, high-dose SPE completely restored Nrf2 to the levels observed in healthy control animals. 

Figure 7.

 

SPE prevented MCE-induced oxidative stress and the subsequent cellular antioxidant response. (A–D) Levels of oxidative stress biomarkers in control rats or rats that received MCE fed with either vehicle or SPE. MCE resulted in increased levels of MDA (A), 8-OH-dG (B), and 4-HNE (C) and in decreased levels of GSH (D). These effects were almost completely prevented or substantially attenuated by SPE in a dose-dependent manner. (E) Representative western blots from retinal homogenates of control rats or rats that received MCE fed with either vehicle or SPE. (F, G) Densitometric analysis of the levels of Nrf2 (F) and HO-1 (G). MCE resulted in increased levels of both Nrf2 and HO-1, effects that were completely prevented or attenuated by SPE in a dose-dependent manner. Data are shown as box plots with minimum to maximum whiskers (n = 10 for each group). ^*P < 0.05 and ^****P < 0.0001 versus control; ^§P < 0.05, ^§§P < 0.01, and ^§§§§P < 0.0001 versus MCE; ^#P < 0.05 and ^##P < 0.01 versus SPE-low (one-way ANOVA followed by the multiple-comparison Tukey's test).

Spearmint Extract Counteracts the MCE-Induced Inflammatory Response in a Dose-Dependent Manner

Inflammatory processes involved in RGC degeneration were analyzed by testing the levels of the active (phosphorylated) form of NF-κB, as one of the master transcriptional regulators of the pro-inflammatory response, and the related levels of pro- and anti-inflammatory cytokines. The western blot shown in Figure 8 revealed that the intracameral injection of MCE resulted in an incremental increase in the phosphorylated form of NF-κB (pNF-κB) (Figs. 8A, 8B), which was paralleled by an increase in pro-inflammatory cytokines IL-6 (Figs. 8A, 8C) and IL-1β (Figs. 8A, 8D) and a significant decrease in the anti-inflammatory cytokine IL-10 (Figs. 8A, 8E). Oral supplementation with SPE exerted a significant dose-dependent inhibition of the pro-inflammatory processes, as demonstrated by the significant decrease in pNF-κB with subsequent reduced levels of IL-6 and IL-1β, and the increase in anti-inflammatory cytokine IL-10 compared to vehicle-treated MCE rats. In this context, the high dose of SPE returned pNF-κB, IL-1β, and IL-10 to healthy control levels. 

Figure 8.

 

SPE prevented the MCE-induced increase in inflammatory response. (A) Representative western blots from retinal homogenates of control rats or rats that received MCE which were fed with either vehicle or SPE. (B–E) Densitometric analysis of the levels of the phosphorylated form of NF-κB (B), IL-6 (C), IL-1β (D), and IL-10 (E). MCE resulted in increased levels of the phosphorylated form of NF-κB, IL-6, and IL-1β and in decreased levels of IL-10. These effects were completely prevented or substantially attenuated by SPE in a dose-dependent manner. Data are shown as box plots with minimum to maximum whiskers (n = 10 for each group). ^*P < 0.05, ^***P < 0.001, and ^****P < 0.0001 versus control; ^§§P < 0.01 and ^§§§§P < 0.0001 versus MCE; ^#P < 0.01, ^##P < 0.01, and ^####P < 0.0001 versus SPE-low (one-way ANOVA followed by the multiple-comparison Tukey's test).

Discussion

Despite many reports describing successful neuroprotection in animal models of glaucoma, the clinical translatability of their results has been extremely limited, possibly because no single model recapitulates all aspects of glaucoma.⁴⁰ However, thanks to basic research, our understanding of the molecular basis of glaucoma pathogenesis is continually increasing, thus opening the door to the development of effective neuroprotective strategies and to their clinical translation. Here, we observed that SPE reduces glaucoma-induced retinal dysfunction acting downstream of the IOP elevation with no effects on IOP itself. In fact, all groups receiving MCE injections exhibited elevated IOP, providing evidence for the validity of the MCE model for reproducing a glaucomatous-like ocular hypertension. Animals exposed to both low- and high-dose SPE showed reduced RGC loss and attenuated functional impairment as a result of the increased IOP. Importantly, the preservation of RGC function and cell density was dose dependent. Exploration of potential underlying mechanisms for the neuroprotection revealed significant, dose-dependent decreases in markers of oxidative stress and inflammation along with the novel finding that oral supplementation with a natural plant extract led to elevated levels of the neurotrophins BDNF and NGF under the glaucomatous conditions. 

Glaucoma provides a model of neurodegeneration in which IOP elevation represents one of the most recurrent and acknowledged risk factors promoting and associated with glaucomatous progression and RGC neurodegeneration.⁵ Glaucoma is understood to be a multifactorial condition influenced by other mechanisms such as oxidative stress, inflammation, and lack of neural trophic support. In addition, progression of neurodegenerative disease is influenced by genetics, environmental risk factors, and nutritional support, evidenced by the fact that some individuals with elevated IOP never develop glaucoma. 

In addition to being commonly monitored as a clinical parameter for the diagnosis and follow-up of glaucoma, IOP elevation currently represents the main target for pharmaceutical treatments attempting to counteract glaucoma progression.⁶ Despite the efficacy of this approach in influencing glaucomatous neurodegeneration, obvious limitations have emerged regarding the actual resolutive effects of treatments targeting IOP reduction and their applicability to IOP-independent glaucomatous neurodegeneration.⁴¹ In effect, IOP elevation is not an overall feature of all glaucoma subgroups because it could manifest later in the progression of the disease or even not occur, as in the case of normotensive glaucoma. These discrepancies would highlight the limitations of current IOP-exclusive treatment strategies for glaucoma management and strengthen the need to seek complementary treatment strategies to counteract IOP-independent neurodegenerative mechanisms. 

Recent advancements in the understanding of neurodegenerative phenomena in glaucoma have pointed to a central role of RGC metabolic imbalance as an early alteration promoting neural complications and cell death in preliminary stages of the disease.⁴² In effect, RGCs are known to display high metabolic needs to fulfill their role in the visual pathway, making them particularly susceptible to metabolic stress and bioenergetic insufficiency.⁴³ Altered metabolism in RGCs would result in mitochondrial dysfunction, thus promoting abnormal surges of reactive oxygen species, ultimately promoting oxidative cell damage.¹¹ Moreover, oxidative stress triggers the activation of inflammatory processes, further promoting cell damage and contributing to the loss of RGC activity and viability.¹⁰ Finally, cell loss in the retina coupled with restricted retrograde transportation in the RGC axon could result in reduced RGC neurotrophin levels.⁴⁴ Therefore, metabolic imbalances of RGCs could contribute to inflammatory, oxidative, and apoptotic damages seen during the early stages of glaucoma. 

As shown in this study, the RGC oxidative, inflammatory and neurotrophic damage typical of glaucomatous conditions is well reproduced by the MCE model of glaucoma. In effect, the increase in IOP promotes selective RGC dysfunction, as demonstrated by the decreased amplitude of the RGC-specific ERG, in agreement with previous studies in rodents.^28,33 These alterations correlate with the loss of RGC density and decreased levels of neurotrophins. In line with the central role played by oxidative stress in glaucomatous phenomena,⁴⁵ MCE-induced losses in RGC activity and viability are associated with an increased oxidative stress paralleled by significant induction of the inflammatory response. 

The emerging roles played by oxidative stress, inflammation, and neurotrophic depletion in the genesis and progression of glaucomatous neurodegeneration have opened up the possibility of using complementary supplementation of bioactive nutrients with antioxidant, anti-inflammatory, and neurotrophic properties, given their beneficial effects on the preservation of RGC activity and viability without treatment invasiveness.⁴⁶ In this respect, the use of SPE as a bioactive ingredient for the complementary nutritional support in the management of glaucoma represents a promising option, given its high content in antioxidant/anti-inflammatory polyphenols. SPE has already displayed significant beneficial effects on age-related cognitive decline in healthy subjects, as well as on neural damages in a mouse model of stroke. These conditions, as well as glaucoma, share increases in oxidative stress and inflammation as common denominators.^18–20 

The present data demonstrate that oral supplementation with SPE starting at 2 weeks before MCE injection diminished (at low dose) or completely prevented (at high dose) IOP-induced neurodegeneration, as evidenced by the maintenance of both RGC density and RGC activity. Together with previous findings demonstrating the efficacy of SPE in the context of a multicomponent formulation,²⁸ the present results point on the effectiveness of SPE, either alone or in combination, in counteracting glaucoma-associated neurodegeneration. In addition to its ameliorative effects on RGC activity and viability, SPE does not induce any changes in the normal outer retinal function, as ERG parameters related to the activity of photoreceptors or second-order retinal neurons are unaltered. Of note, SPE exerts neuroprotection independently from any hypotensive effect, suggesting that it acts on downstream effectors that are affected by an increase in IOP. In this respect, because SPE is rich in RA and other polyphenol compounds such as salvianolic and lithospermic acid, which are known for their anti-inflammatory and antioxidant properties,^47,48 the effects of SPE on NF-κB activity and inflammatory cytokines, as well as on markers of oxidative stress, could be expected. However, the finding that GSH levels may be completely preserved by SPE is noteworthy, as it indicates that this polyphenol-rich ingredient may restore the non-enzymatic antioxidant endogenous defenses, which act as rapid inactivators of radicals and oxidants. Nevertheless, the novel finding that SPE may attenuate the reduced production or availability of neurotrophins seen in the glaucomatous condition is of particular importance. In fact, the lack of neurotrophin supply to RGCs is a mechanism common to different retinal neurodegenerative diseases^13,49 and the use of neurotrophins to counteract retinal degeneration has been widely discussed.⁵⁰ Furthermore, neurotrophins are key for neuronal cell survival and synaptic plasticity in the optical system and are involved in learning and memory processes.^31,32 The metabolic and neurotrophic imbalances in retinal tissues can be related to the morphological and functional changes along the visual pathway and in numerous brain areas outside the visual pathway known to affect the CNS in glaucoma.⁷ 

The present finding, therefore, may open the door for the use of SPE supplementation as nutritional support in patients with glaucoma, as well as for patients suffering from other neurodegenerative retinal conditions such as retinitis pigmentosa, age-related macular degeneration, or optic neuritis.^51–53 Moreover, the effect of SPE in the preservation of neurotrophin levels under neural stress conditions may further expand the suitability of including this ingredient in nutritional support dedicated to brain disorders, in which altered neurotrophism classically drives neurovascular breakout.⁵⁴ Although this is currently a hypothesis that requires specific investigations, the effects of SPE previously observed on the blood–brain barrier¹⁸ and in models of stroke¹⁹ may point on this direction. Nevertheless, the present results regarding SPE effects on neurotrophins provide an additional proposed mechanism for the cognitive function benefits already observed in clinical trials with SPE.^23,25 

If viewed from a translational perspective, the present study has potential limitations that are linked to the model used here. In the rat, IOP-induced RGC damage occurs over a short period of time, whereas the same damage in humans requires years to manifest. In addition, because MCE blocks aqueous humor outflow, the model used here could be related to primary angle-closure glaucoma, which affects only a quarter of the glaucoma population despite being the cause of about 50% of glaucoma-related blindness.⁵⁵ Another potential limitation is that here we observed a neuroprotective effect of SPE starting with a prophylactic approach; therefore, whether SPE may produce similar results when given only in a therapeutic setting has yet to be proven. Furthermore, it is not obvious what the best time window is in which to start SPE administration in humans not affected by glaucoma in order to obtain effects similar to those observed here. The effects of SPE on the mechanisms underpinning glaucoma development in the MCE rat model are summarized in Figure 9. 

Figure 9.

 

Schematic representation of SPE effects on glaucoma pathophysiological mechanisms. (A) In glaucoma, several factors coincide, either increasing IOP or not, to create an imbalance in the metabolic activity of RGCs. The increase in oxidative stress and inflammation, two processes that mutually interact one another, as well as the consequent reduction in RGC trophism, lead to a reduction in RGC activity and viability. (B) SPE likely exerts an antioxidant effect due to the scavenging properties of its main component, RA, and other spearmint polyphenols, thus inhibiting the IOP-induced increases in oxidative stress. The inhibition of oxidative processes consequently impacts the inflammatory response and reduces inflammation, thus decreasing the RGC metabolic alterations that typically occur in both hypertensive and normotensive glaucoma. The restoration of the metabolic balance is reflected by the enhancement of RGC neural trophism which correlates with the preservation of both cell activity and viability.

Conclusions

The present findings demonstrate the efficacy of SPE, a plant-based clinically studied nootropic ingredient, as a promising nutritional support in modulating neuroprotective mechanisms relevant for the management of glaucoma symptoms. The data emphasize the IOP-independent effects of SPE in reducing markers of oxidative stress and inflammation and promoting neurotrophism, thus highlighting the possibility of its application, either alone or in combination with other ingredients (as already reported in the literature), as a complementary approach in support of hypotensive treatments aimed at optimizing the management of glaucoma. We are aware that an important limitation in the clinical management of glaucoma is establishing the actual risk of glaucoma development in patients, as the diagnosis emerges in relatively advanced stages of the disease. However, a growing body of evidence has recently highlighted the possibility of identifying, over the progression of glaucoma in patients, the so-called “critical period.” It would correspond to oxidative stress- and inflammatory-related RGC dyshomeostasis preceding cell loss that could be detected with the use of sensitive electrophysiological tests such as PERG.⁵⁶ 

Acknowledgments

The authors thank Tiziana Cintio for her assistance with animal housing and care. These data were presented (E-abstract 3880242) at the ARVO meeting held in New Orleans, LA, in April 2023. 

Supported by a grant from Kemin Foods, L.C. (Des Moines, IA) to MDM. Kemin Foods, L.C., had no direct role in the collection, analyses, or interpretation of the data. 

Disclosure: R. Amato, None; A. Canovai, None; A. Melecchi, None; S. Maci, (E); F. Quintela, (E); B.A. Fonseca, (E); M. Cammalleri, None; M. Dal Monte, (F)