Effects of oral Mirtogenol on retinal ganglion cell apoptosis index and intraocular pressure in the Wistar glaucoma model

ABSTRACT

The purpose of this research is to determine how Mirtogenol affects intraocular pressure (IOP) and retinal ganglion cells (RGCs) of apoptosis index in Wistar glaucoma models, as well as the relationship between IOP and RGC apoptosis index. Twelve Wistar glaucoma models were divided into two groups for experimental research with a pretest–posttest and posttest-only. The treatment group got oral administration of Mirtogenol 12.3 mg twice a day for 2 weeks, whereas the control group received a placebo in the same way. Apoptotic index and IOP were evaluated both before and after the intervention. A parametric independent t-test was used to determine the difference between groups, and a parametric paired t-test was used to determine the difference within groups. The results showed that the RGC apoptosis index in treatment groups was considerably less when compared to control groups (P < 0.001). In the treatment group, the IOP is decreased compared to the control group (mean difference: −12.67 ± 3.79 vs. 0.69 ± 4.64, respectively, P = 0.002). A significant and solid correlation was found between IOP and RGC apoptosis index (R = 0.884, P < 0.001). Thus, Mirtogenol supplementation is expected to be used to prevent glaucoma progression.

INTRODUCTION

Retinal ganglion cell (RGC) death and frequent visual field abnormalities are symptoms of glaucoma. According to Tham et al., global glaucoma incidence is expected to increase to 76 million by age 40–80 by 2020 and 111.8 million by 2040, with a higher prevalence in African and Asian races.[1] Indonesia faced similar challenges with data from National Basic Health Research (Riset Kesehatan Dasar/RISKESDAS) in 2007 showed that 4.6 out of 1000 Indonesians had been diagnosed with glaucoma.[2] The risk of glaucomatous neuropathy is influenced by inter-related factors, such as high intraocular pressure (IOP), vascular dysregulation inflammation, oxidative stress, and changes in the extracellular matrix. The high IOP caused oxidative stress and ischemic conditions in the papillary nerve cell that will trigger the molecular and functional change of RGC axons and promote apoptosis.[3,4,5,6,7]

High IOP is a reduced risk factor that can be managed with medication and surgery. The current treatment aims to maintain visual function by lowering IOP until it reaches the target pressure. However, this approach rarely stops the irreversible damage process. Emerging evidence indicates that the pathogenesis of glaucoma is influenced by complex systems such as free radicals, inflammation, and vascular disorders.[8] The American Academy of Ophthalmology advises treating glaucoma by lowering the goal IOP by 25% from the baseline IOP. However, controlling IOP does not always stop optic nerve damage.[9] Hence, additional therapies and new therapeutic options for glaucoma are urgently needed.[10]

Mirtogenol is made from French maritime pine bark (Pycnogenol) extract and standardized bilberry extract (Mirtoselect). It enhances endothelial function by increasing endothelial nitric oxide synthase activity, increasing endothelial nitric oxide production. Mirtogenol also counteracts the hyperpermeability of ciliary capillaries and exerts antioxidant effects. Mirtogenol administration is expected to have a protective effect of preventing RGC apoptosis and reducing IOP to prevent glaucoma progression.[11]

Steigerwalt et al. showed that 3-month Mirtogenol administration could reduce IOP and further increase ocular blood flow after 6 months of usage.[12] Further research confirmed the IOP reduction after individual Mirtogenol or latanoprost administration or a combination of Mirtogenol and latanoprost administration, with the highest IOP reduction, is obtained in Mirtogenol and latanoprost combination therapy.[11]

In previous studies, the effect of Mirtogenol on IOP reduction has been discussed, but there has been no explanation of the effect of Mirtogenol on the RGC apoptosis index. Thus, this study aimed to observe Mirtogenol effect on IOP and RGC apoptosis index and to investigate the correlations between IOP and RGC apoptosis index of Wistar glaucoma models. The apoptosis index was assessed using the Allred score based on the density and intensity of RCG cells in the retinal cell layer after immunohistochemically staining using rabbit polyclonal caspase 3 antibodies (EPR16888) and Abcam to detect caspase 3 activation which the process should not be done on humans because it violates ethics.

Wistar/rat glaucoma model was developed to study the expression of myocilin. The topical injection of dexamethasone produces this model. Rats have several advantages, such as having the anterior chamber’s anatomical and developmental traits with humans, particularly in the aqueous outflow pathway, anticipating the imitation changes in humans, and employing in huge quantities. Moreover, rats are easier to keep in laboratories, allowing for genetic manipulation.[13]

MATERIALS AND METHODS

This was an experimental study with a pretest–posttest randomized controlled group design for IOP and a posttest randomized controlled group design for the apoptosis index in the Wistar glaucoma model. The rats were housed at the Experimental Animal Laboratory, Faculty of Medicine, Universitas Diponegoro, also used as an experimental site from March to May 2019. The samples used were male Wistar rats aged 6–8 weeks with IOP ≥ 30.1 mmHg after episcleral vein cauterization (EVC) with a body weight of 150–200 g. EVC creates a glaucoma model with high IOP. However, the long-term retinal harm in this glaucoma paradigm has not been precisely measured.[14] Those rats that appeared active during the adaptation period and did not have eye anatomic abnormalities were recruited as study samples. Rats with infectious diseases, aggressive behavior, illness, and death were excluded from the study. Ethics approval was obtained from the Research Ethics Commission, Health Faculty of Medicine, University of Diponegoro (Reference Number: 15/EC/H/FK-UNDIP/III/2019).

The samples were randomly allocated to the treatment group (P) and control group (K), comprising six rats for each group. Oral Mirtogenol was given to the treatment group by calculating the dose using the human equivalent dose formula based on body surface area. The dose of Mirtogenol in humans was 120 mg Optimax for G, produced by PT. Ferron Par Pharmaceuticals. The group was treated with 12.3 mg/kg oral Mirtogenol every 12 h for 2 weeks. The control group (K) was given equates as a placebo with a similar administration manner.

The sample size calculation is based on the provisions of the WHO, namely, at least five rats in each group. If that counts, the probability of dropping out is 10%. Hence, the sample size for each group is:

Each group had a sample size of six rats for 12 samples for the two categories. The rats were accustomed to the same environment for 2 weeks before being used in the research. The rats were given unlimited access to food and water during the adaptation time. The rats were then randomly split into groups, Group K and Group P, each with six rats.

The primary measured outcome for this study was IOP and RGC apoptosis index. IOP was measured three times before and after the intervention using Reichert XL to open. Previously, the instrument was calibrated and measured by the same measuring officer. Pre-IOP was IOP after EVC before treatment (administration of oral Mirtogenol and placebo). In comparison, post-IOP was IOP after treatment (administration of Mirtogenol oral and placebo) and measured after 2 weeks. The apoptosis index was assessed using the Allred score based on the density and intensity of RCG cells in the retinal cell layer after immunohistochemically staining using rabbit polyclonal caspase-3 antibodies (EPR16888) and Abcam to detect caspase-3 activation.

The Shapiro–Wilk test assessed data distribution to observe Mirtogenol effect on IOP and RGC apoptosis index. Normal data distribution was obtained for all variables except the initial IOP in the treatment group. The intragroup difference test used the nonparametric Wilcoxon signed-rank test, and the intra-control group difference test used the parametric paired t-test. The posttreatment IOP difference test (between the treatment and control groups) used a parametric independent t-test and a different test for the RGC apoptosis index using a parametric independent t-test. The correlations between IOP and RGC apoptosis index were measured using the Pearson correlation test, with a significant correlation at P < 0.001. If the data were normally distributed, a parametric independent test was used to evaluate the difference between groups, and a parametric paired t-test was used to measure the intragroup difference. If the data were abnormally distributed, the difference between groups was measured using the Mann–Whitney test and Wilcoxon signed rank for the intragroup difference. The results were considered statistically significant if P < 0.05.

RESULT AND DISCUSSION

Figures 1, 2 and Table 1 illustrate the mean, standard deviation, and statistical analysis in each glaucoma model group. Significant IOP reduction was seen in the treatment group compared to the control group (−12.67 ± 3.79; P = 0.002 vs. −0.69 ± 4.64; P = 0.613, respectively). It also found a statistically significant difference (P < 0.001) in the postintervention IOP between K and P groups (P < 0.001).

Figure 1.

Boxplot diagram of initial mean IOP in treatment and control group. IOP: Intraocular pressure

Figure 2.

Boxplot diagram of final mean IOP in treatment and control groups. IOP: Intraocular pressure

Table 1.

Mean intraocular pressure of Wistar glaucoma model

	Group		P
	Treatment	Control
Initial IOP (mmHg)	38.36±4.31	38.22±3.77	0.885a
Final IOP (mmHg)	25.69±2.21	37.52±4.26	<0.001b,*
P	0.002c,*	0.613d
Difference	−12.67±3.79	−0.69±4.64

^aMann–Whitney test, ^bIndependent t-test, ^cWilcoxon signed-rank test, ^dPaired t-test. Significant, *Significant difference (P < 0.001). IOP: Intraocular pressure

Table 2 shows the comparison results of the RGC cell apoptosis index between Group P and Group K. It is found a statistically significant difference between the two groups (P < 0.001).

Table 2.

Mean retinal ganglion cell apoptosis index based on the Allred score criteria in treatment and control groups

Group	Apoptosis index (mean±SD)	P
Treatment	2.58±0.37	<0.001∫,*
Control	6.27±0.64

^∫Independent t-test, significant, *Significant difference (P < 0.001). SD: Standard deviation

Figure 3 illustrates mean RGC cell apoptosis index in control and treatment group. Pearson correlation test between IOP and the apoptotic index [Table 3] found a positive and very strong correlation between IOP and the RGC apoptotic index (correlation coefficient 0.884) with a significant correlation (P < 0.001).

Figure 3.

Boxplot diagram of mean RGC cell apoptosis index in the control and treatment group. IOP: Intraocular pressure, RGC: Retinal ganglion cells

Table 3.

Correlation between intraocular pressure and retinal ganglion cell apoptosis index

Variable	Mean±SD	P	R	Notes
Apoptosis index	4.43±1.95	<0.001*,a	0.884	Significant, positive, very strong
IOP	31.61±6.89

*Significant, ^aPearson’s correlation test, Significant correlation (P<0.001). SD: Standard deviation, IOP: Intraocular pressure

This research demonstrated that oral Mirtogenol administered at 12.3 mg/kg every 12 h for 2 weeks could lower mean IOP in Wistar glaucoma models by 12.67 mmHg or 33% compared to the control group (no significant IOP reduction was found). The results of this study follow several previous studies. A pilot study by Steigerwalt et al. showed that increased ocular blood flow might help prevent glaucoma. By regulating IOP and enhancing ocular blood flow, Mirtogenol may be a secure preventative strategy for reducing the chance of acquiring symptoms of glaucoma.[11] Further research by Steigerwalt et al. confirmed that although it takes longer, Mirtogenol reduced patients’ increased IOP almost as effectively as latanoprost.[12] Both treatments worked better to decrease IOP, and the combined effects improved retinal blood flow.

A comparison of the supplement’s and latanoprost’s separate effects on decreasing IOP with the combination therapy reveals an additive impact: Mirtogenol primarily impacts the vascular response involved in ocular hypertension by restoring ciliary body capillary filtration. Mirtogenol can also counteract the hyperpermeability of ciliary capillaries and improve central retinal artery blood flow and endothelial function; hence, it causes a decrease in intraocular blood pressure.[11] This study also showed that Mirtogenol therapy could lower the RGC apoptosis index compared to the control group (2.58 ± 0.37 vs. 6.27 ± 0.64, P < 0.001). This result shows that Mirtogenol can reduce RGC apoptosis. In glaucoma, the RGC apoptotic mechanism is thought to be induced by various stimuli, including ischemia, oxidative stress, and increased glutamate levels. Mirtogenol supplementation could reduce plasma free radicals, creating a protection system against increased oxidative stress and vasospasm. Two Mirtogenol ingredients (Bilberry and Pycnogenol) are potent antioxidants.[15,16,17] Previous studies have shown that Mirtoselect^® can enhance several microcirculation and perfusion parameters, whereas Pycnogenol can increase endothelial vasodilation and decrease systolic blood pressure by inducing vascular vasospasm, hence improving optic nerve hypoxic conditions.[18,19]

This study also revealed a positive correlation between IOP and RGC. According to previous studies, IOP reduction could enhance axoplasmic flow. It is necessary for transporting nutrients, proteins, and intracellular glycolysis, enhancing RGC metabolism. Furthermore, lower IOP enabled the retrograde movement of neurotrophins, which are essential for RGC development, regeneration, and differentiation. Reduced IOP will also diminish astrocytic interleukin release (e.g., tumor necrosis factor), improving RGC function.[20]

CONCLUSIONS

This study aims to find out how Mitrogenol influences IOP and RGC apoptosis index and its correlation in Wistar glaucoma. The study reveals that oral Mirtogenol supplementation can significantly reduce the Wistar glaucoma model’s IOP and RGC apoptosis index. The correlation between IOP and RGC also showed a significant relationship. Thus, Mirtogenol supplementation is expected to prevent glaucoma progression by contributing as supplementary management to reach a normal IOP and decrease the apoptosis index in RGC.

The effects of Mirtogenol on the Wistar glaucoma model are being examined for the first time in this research. It has limitation on the use of one dose and one route of administration of Mirtogenol, which cannot compare dose-dependent and administration efficacy. This study also cannot explain whether the apoptotic index decrement was solely due to IOP reduction or whether there is a direct effect of Mirtogenol in the apoptotic pathway. Therefore, for further study, those two problems can be investigated.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.