The evaluation of the effect of tafluprost on the intraocular pressure of healthy male guinea pigs under different light‐and‐darkness regimes

Abstract Background Ocular hypertension is one of the most underdiagnosed ocular abnormalities among guinea pigs around the world. Objectives The current study investigates the effect of 0.0015% preservative‐free tafluprost ophthalmic solution (Zioptan) on the intraocular pressure of 16 healthy male guinea pigs (Cavia porcellus) under different light/darkness regimes. Methods All guinea pigs received a single drop of tafluprost at 5:30 in the right eye, whereas the contralateral eyes served as control to receive a placebo. Then, the animals were randomly divided into two groups; group A was exposed to light, whereas group B was placed in darkness from 5:30 to 18:00. Rebound tonometry (TonoVet) was instrumented to measure IOP values at 5:30 (baseline), 6:00, 7:00, 8:00, 9:00 and then every 3 h until 18:00. Results The maximum IOP reduction associated with tafluprost was observed at 6:00 by −1.4 ± 1.1 mmHg (p‐value = 0.026) and −2.5 ± 1.2 mmHg (p‐value = 0.011) in group A and B, respectively (repeated measure ANOVA test). There was a significant difference between the mean right and left eye IOP values in both groups at 5:30, 6:00, 7:00 and 8:00 (p‐value <0.05), which was greater in amount in group B compared to group A due to the effect of darkness on IOP reduction. Conclusions It is suggested that the variations of IOP in different light/dark conditions be taken into consideration when applying ocular hypotensive agents on guinea pigs’ eyes.


INTRODUCTION
Glaucoma is one of the driving causes of visual deficiency around the world. Due to the progressive nature of the ailment and the difficulty of ocular disease diagnosis in animal species, timely diagnosis and proper treatment are necessary for vision loss prevention (Komáromy et al., 2019;Weinreb et al., 2014). Although guinea pigs are widely kept as pet companions and laboratory animals, there have been a few investigations on the prevalence of ocular diseases in this specie (Williams & Sullivan, 2010).
One such study reveals that in a population of 1000 guinea pigs without any apparent changes in health and behaviour, 45% of them were diagnosed with ocular conditions associated with glaucoma, namely cataracts (21%) and lens lesions (17%) (Williams & Sullivan, 2010). These animals often conceal their disease symptoms instinctively in the wild being a prey specie not to steer the attention of predator species (Edis & Pellett, 2018). As a result, several chronic diseases of this specie may remain underdiagnosed and progress into advanced stages at the time of veterinary visits, such as prolonged ocular hypertension as a major glaucoma risk factor (Komáromy et al., 2019).
The reduction of the IOP by topical ophthalmic solutions is currently the mainstay of glaucoma therapy at the initial stages of the disease (Ruangvaravate et al., 2020). However, the interspecies differences among mammalian species in aqueous humour production and drainage pathways, as well as metabolic and anatomical differences, are responsible for the different efficacy of ocular hypotensive medications (Ofri, 2002;Papadia et al., 2011).
Moreover, it is believed that the daily cycles of light and darkness among other environmental factors can influence the diurnal pattern of IOP fluctuations (Buijs & Kalsbeek, 2001;Cahill & Menaker, 1989). In this study, we aim to evaluate the effect of tafluprost as a novel ocular hypotensive solution regarding the effect of light and darkness on IOP in the guinea pig. Two weeks before the study, all animals were placed in a laboratory room with no windows to recover from shipping-related stress and to acclimatize to the new light/darkness regime, which consisted of 12 h of light followed by 12 h of darkness (Devlin & Kay, 2001;Reppert & Weaver, 2002). The application of light was from above the animals' heads at all times during the light phase provided by a white 40-W LED lamp accompanied by a yellow 9-W LED lamp. To avoid any IOP irregularities that would affect the study results, all IOP measurements during the dark phase were performed in the same room using dim red light provided from the room corner (Aihara et al., 2003;Liu et al., 1999). A same diet consisting of fresh fruits and vegetables and guinea pig commercial pallets was provided for all animals as nutrition is believed to influence IOP due to its effect on metabolism (Green et al., 2008). There was no food restriction for the animals, and water was ad liberum at all times before and during the experiment.

MATERIALS AND METHODS
White pine shavings were used to cover the floor of individual boxes Then, all animals in group A were exposed to 12 + 0.5 h of light from 5:30 to 18:00, whereas those in group B were exposed to 12 + 0.5 h of darkness. After 30 min, the next IOP measurement was done in groups A and B on both eyes at 6:00, and then every hour at 7:00, 8:00 and 9:00. For the rest of the 12-h period of the experiment, the IOP measurements were performed every 3 h (at 12:00, 15:00 and 18:00) to evaluate the pattern of IOP fluctuations in both eyes. All IOP measurements and data collections were performed by a single experimenter to eliminate individual bias.

Statistical analysis
All the statistical analyses were performed by the SPSS statistical

RESULTS
The baseline right and left eye mean ± SD IOP values (at 5:30) were 9.0 ± 1.8 and 8.6 ± 1.2 mmHg in group A and 9.9 ± 1.9 and 9.3 ± 1.4 mmHg in group B, respectively (Table 1).
To evaluate the IOP-lowering effect of tafluprost, the mean IOP values at each time during the experiment were analysed by repeated measure ANOVA, which is presented in  (Liu, 2013).
There was a significant reduction in the IOP values of both eyes in groups A and B post instillation of tafluprost at all times because of the effect of ophthalmic solution (right eyes) and systemic absorption (left eyes), except at 9:00 for the right eye (p-value = 0.059) and at 15:00 for the left eye (p-value = 0.067) in group A, which is attributable to type 2 error due to small sample size.

DISCUSSION
To this day, it is the first experiment performed on guinea pigs to study the effect of tafluprost and light/darkness conditions simultaneously.
According to the results of IOP measurements at baseline, the mean right eye IOP was slightly higher than the left eyes in both groups, which is attributable to the order of IOP measurements because the right eye was chosen first for all IOP recordings during the experiment (Pekmezci et al., 2011). A study by Pekmezci in 2011 demonstrated the importance of IOP measurement order, where the first measured eye always had higher IOP values, mostly due to ocular squeezing (Pekmezci et al., 2011).
Tafluprost as a topical IOP reducing ophthalmic solution was first introduced to the market in 2008 and has shown to be effective on IOP reduction in humans, dogs, monkeys and mice (Liu & Mao, 2013;Ota et al., 2007;Shokoohimand et al., 2020;Takagi et al., 2004). This ophthalmic solution was later approved in 2012 by the Food and Drug Association for use in humans for open-angle glaucoma and ocular hypertension treatment (Ruangvaravate et al., 2020).
Previous studies on humans with normal ocular tension demonstrated that IOP was reduced significantly post instillation of tafluprost by −4.0 ± 1.7 mmHg compared to the placebo group (−1.4 ± 1.8 mmHg) after 4 weeks of therapy (p-value <0.001) (Kuwayama & Komemushi, 2010). In a study conducted by Akaishi et al. (2009) in mice, IOP was significantly reduced after 1-3 h by −2.7 ± 0.6, −4.1 ± 1.3 and −5.7 ± 0.5 mmHg, respectively after tafluprost and timolol conjunctive therapy. In another study on the effect of tafluprost (single drop) on canine eyes, IOP was significantly reduced up to 6 mmHg (39% reduction) compared to the control group after 8 h (Kwak et al., 2017).
Although there was a significant difference between the right and left eye IOP values in both groups post instillation of tafluprost (except at 18:00 in group A, and also 9:00 and 12:00 in group B, Table 3), the reductions of the left eye IOP values were less than the right eye and occurred with a delay, which can be attributed to the systemic absorption of the prostaglandin analogue ophthalmic solutions.
A study by Akaishi et al. (2010) in rabbits showed that although a part of the IOP-reducing effect of prostaglandin analogues is due to the FP receptors, the stimulation of endogenous prostaglandins in response to FP stimulation is also suggested to contribute to the systemic absorption of these drugs. In another study by Ota et al. (2007) in mice, the stimulation of prostanoid EP3 receptor by FP receptor-mediated prostaglandin production is believed to be respon- which in humans are a function of suprachiasmatic nuclei located in the anterior hypothalamus (Halberg, 1959;Moore et al., 2002;Reiss et al., 1984;Smolensky & Haus, 2001). Although the activity of the internal pacemaker can happen in the absence of external stimuli, many environmental factors contribute to its function, most importantly, the daily cycles of light and darkness (Buijs & Kalsbeek, 2001;Cahill & Menaker, 1989).
Although there have been several studies on the diurnal rhythms of IOP in different animal species, there are some significant variations among them, indicating a direct relationship between higher IOP values and activity/awakening phases (Del Sole et al., 2007). A study on the effect of circadian rhythm on daily IOP fluctuations in rabbits (12 h light/12 h darkness) revealed that IOP values increased by 10 mmHg (Alcon Applanation Pneumography) with the onset of darkness, whereas decreased by the same amount with the onset of light (Rowland et al., 1981). A similar study on rats evaluated the diurnal pattern of IOP fluctuations, in which IOP values, measured by TonoPen XL tonometer, first decreased and then increased in light (19.3 ± 1.9 mmHg) and dark (31.3 ± 1.3 mmHg) conditions, respectively (Moore et al., 1996). With regard to previous literature, in nocturnal species such as cats, the maximum IOP values were recorded at night (Del Sole et al., 2007), whereas in both rhesus macaques and beagle dogs, IOP was highest in the early morning (Bito et al., 1979;Chen et al., 1980 Prostaglandin analogue ophthalmic solutions, such as tafluprost, are considered safe and convenient when applied once daily. Even though several local side effects (conjunctival hyperemia, mild irritation of the eye and pigmentation of eyelid, periocular skin and iris) have been reported in prolonged use of tafluprost in less than 7.7% of patients (Feldman, 2003;Honrubia et al., 2009;Kuwayama & Nomura, 2014;Papadia et al., 2011), no local side effects were observed in the guinea pigs post instillation of a single drop of tafluprost during the 12 + 0.5 h of the experiment.

CONCLUSIONS
The results of the current study demonstrate that a variation in the IOP values of the guinea pigs post instillation of tafluprost is present between the two groups, which is attributable to the carefully monitored conditions of light and darkness. Therefore, IOP fluctuations during the day and night are important parameters to be considered in ocular hypertension treatment.

ACKNOWLEDGEMENTS
We would like to thank the Faculty of Specialized Veterinary Sciences of the Science and Research Branch of Islamic Azad University (IAU), Tehran, Iran, for their approval and support in conducting this investigation.

CONFLICT OF INTERESTS
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. No financial or conflict of interest that might have biased the work have been declared.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

ETHICS STATEMENT
All guidelines regarding the work with laboratory animals have been observed during the experiment and animals handling.