Dual-Mode Gold Nanoparticle-Based Method for Early Detection of Acanthamoeba.

Acanthamoeba keratitis is an aggressive and rapidly progressing ocular pathology whose main risk factor is the use of contact lenses. An early and differential diagnosis is considered the main factor to prevent the progression and improve the prognosis of the pathology. However, current diagnosis techniques require time, complex and costly materials making an early diagnosis challenging. Thus, there is a need for fast, accessible, and accurate methods for Acanthamoeba detection by practitioners for timely and suitable treatment and even for contact lens user as preventive diagnosis. Here, we developed a dual-mode colorimetric-based method for fast, visual, and accurate detection of Acanthamoeba using gold nanoparticles (AuNPs). For this strategy, AuNPs were functionalized with thiolated probes and the presence of target Acanthamoeba genomic sequences, produce a colorimetric change from red to purple. This approach allows the detection of 0.02 and 0.009 µM of the unamplified Acanthamoeba genome by the naked eye in less than 20 min and by color analysis using a smartphone. Additionally, real samples were successfully analyzed showing the potential of the technology considering the lack of point-of-care tools that are mostly needed.

Contact Lenses Loaded with Melatonin Analogs: A Promising Therapeutic Tool against Dry Eye Disease.

Melatonin analogs topically administered evoke a potent tear secretagogue effect in rabbits. This route of drug administration requires high drug concentration and frequent dosing due to its reduced ocular surface retention. Therefore, contact lenses (CLs) have emerged as an alternative drug-delivery system that prolongs drug retention in the cornea, improving its therapeutic performance. This study explores the in vitro ability of five commercially available hydrogel CLs to act as a delivery system for melatonin analogs and the in vivo secretagogue effect of melatonin analog-loaded CLs. We soaked CLs with melatonin or melatonin analog solutions (1 mM) for 12 h. Spectroscopic assays showed that IIK7-loaded CLs led to the inadequate delivery of this compound. Conventional hydrogel lenses loaded with agomelatine released more agomelatine than silicone ones (16-33% more). In contrast, the CLs of silicone materials are more effective as a delivery system of 5-MCA-NAT than CLs of conventional materials (24-29%). The adaptation of CLs loaded with agomelatine or 5-MCA-NAT in rabbits triggered a higher tear secretion than the corresponding eye drops (78% and 59% more, respectively). These data suggest that CLs preloaded with melatonin analogs could be an adequate strategy to combat aqueous tear deficient dry eye disease.

Therapeutic potential of topical administration of siRNAs against HIF-1α for corneal neovascularization.

Given the implications of the problem of neovascularization on ocular health, as well as the growth in the number of cases, the purpose of the present study has been testing the efficacy of siRNAs (small interfering RNA) designed to silence Hypoxia Inducible Factor -1α (HIF-1α) and to demonstrate that their use stops neovascularization in a model of corneal burn. Corneal wounds in the limbic zone were made in the eyes of New Zealand white rabbits. Topical applications of siRNAs were done the next day to the wound for four consecutive days and eyes were examined with a slit lamp. Evaluation of neovascularization progress was done by analyzing images by ImageJTM and to determine the neovascular area in Matlab ® was used. At the same time, a rabbit corneal cell line was used for in vitro study of hypoxia exposure and Western blot analysis of the cell's extracts were done. Under normal cell culture oxygenation, the expression of HIF-1α was lower than that observed under hypoxic conditions. After 2 h of hypoxia, there was a significant increase in the HIF-1α expression, effect that was maintained up to 6 h. The increased in HIF-1α was mimicked by a cell permeable prolyl-4-hydroxylase inhibitor. Cobalt chloride showed no capacity to increase HIF-1α in vitro. The effect of three different siRNA on HIF-1α was tested after 4 h of hypoxia. siRNA#1 was able to silence 80% of HIF-1α expression, siRNA#2 and siRNA#3 reduce the expression in 45% and 40% respectively. In addition, the three siRNA were tested in a corneal model of neovascularization. scrambledsiRNA#2 was the most effective inhibitor of blood vessel production, followed by siRNA#3 and siRNA#1. Compared to the scrambled siRNA (100% of blood vessel generation), siRNA#2 blocked the presence of blood vessels by 83 ± 2%, siRNA#3 inhibited 45 ± 7% and siRNA#1 only inhibited 18 ± 5%. The necessary time to observe the 50% of effect showed values of NV50 of 10.2 ± 2.4 days for the scrambled siRNA, 9.1 ± 1.4 for siRNA#1, 6.5 ± 1.85 for siRNA#2 and 4.8 ± 1.8 days for siRNA#3. In conclusion, the topical application of siRNA towards HIF-1α seems to be an effective and reliable method to stop neovascularization.

Neutrophils and neutrophil extracellular trap components: Emerging biomarkers and therapeutic targets for age-related eye diseases.

Age-related eye diseases, including dry eye, glaucoma, age-related macular degeneration, and diabetic retinopathy, represent a major global health issue based on their increasing prevalence and disabling action. Unraveling the molecular mechanisms underlying these diseases will provide novel opportunities to reduce the burden of age-related eye diseases and improve eye health, contributing to sustainable development goals achievement. The impairment of neutrophil extracellular traps formation/degradation processes seems to be one of these mechanisms. These traps formed by a meshwork of DNA and neutrophil cytosolic granule proteins may exacerbate the inflammatory response promoting chronic inflammation, a pivotal cause of age-related diseases. In this review, we describe current findings that suggest the role of neutrophils and their traps in the pathogenesis of the above-mentioned age-related eye diseases. Furthermore, we discuss why these cells and their constituents could be biomarkers and therapeutic targets for dry eye, glaucoma, age-related macular degeneration, and diabetic retinopathy. We also examine the therapeutic potential of some neutrophil function modulators and provide several recommendations for future research in age-related eye diseases.

Visual function, ocular surface integrity and symptomatology of a new extended depth-of-focus and a conventional multifocal contact lens.

PURPOSE: To evaluate visual function, ocular surface integrity and dry eye symptoms with an extended depth-of-focus (EDOF) design and a conventional multifocal (MF) contact lens (CL) after 15 days of wear. METHODS: A crossover single mask randomised clinical trial was conducted including 30 presbyopes who used an EDOF and a conventional MF CL (Biofinity MF) for 15 days each. Defocus curves, depth-of-focus range, contrast sensitivity (CS) under photopic and mesopic conditions (with and without glare) and subjective perception of halos and glare were evaluated. The ocular surface was evaluated through non-invasive Keratograph tear breakup time (NIKBUT), averaged tear breakup time (NIKBUT-avg), tear meniscus height (TMH), bulbar and limbal redness, and conjunctival and corneal staining. Dry eye symptoms were assessed with the OSDI questionnaire. RESULTS: No statistically significant differences were found for defocus curves or depth-of-focus between the two CLs (both p > 0.05). Subjective perception of halos and glare was not significantly different between CLs. Statistically significant differences were observed for CS under mesopic conditions for low spatial frequencies (p = 0.008). None of the CL produced significant changes in NIKBUT, NIKBUT-avg, TMH or redness. No change in conjunctival staining was observed in 76.7 % and 73.3 % of participants for EDOF and Biofinity MF, respectively. No change in corneal staining was observed in 86.7 % and 83.3 % of participants for EDOF and Biofinity MF, respectively. No changes were observed in the symptomatology measured with OSDI questionnaire (p > 0.05). CONCLUSIONS: Both CL for presbyopia offer good visual quality, preserve the ocular surface integrity and provide the patient with similar symptomatology levels after 15 days of lens wear.

Impact of contact lens wear on NLRP3 gene expression: Implications for ocular frailty in middle-aged adults.

The inflammatory process plays a crucial role in frailty syndrome, which can appear in middle age and is associated with a poor health outcome. Consequently, gerontologists recommend screening inflammatory biomarkers in middle-aged adults to detect frailty and, therefore, prevent chronic diseases and mortality. External factors could be a risk factor for frailty because they can generate and extend the inflammatory process. For these reasons, we analysed the effect of long-term contact lens wear on mRNA level of genes linked to inflammation (IL-6, NLRP3, NK1R, CD73, MUC16 and TRPV1 genes) in conjunctival cells of middle-aged individuals, by quantitative PCR. Middle-aged contact lens wearers presented a significant increase of NLRP3 and MUC16 mRNA level as well as a decrease of CD73 mRNA level, in comparison with non-contact lens wearers. Additionally, we checked for a potential correlation between these transcript levels and clinical changes of the participants' ocular surface. Unlike molecular analysis, clinical examination fails to detect inflammation in contact lens wearers. These data suggest that long-term contact lens wear could trigger an inflammatory response in middle age orchestrated by NLRP3 inflammasome and modulated by CD73 and MUC16 proteins. Further studies are needed to confirm our gene expression findings at the protein level as well as to investigate the potential role of long-term CL wear in the onset of ocular frailty.

Optimization of a Rabbit Dry Eye Model Induced by Topical Instillation of Benzalkonium Chloride.

PURPOSE: To optimize a rabbit dry eye model induced by topical instillation of benzalkonium chloride (BAC), reduce the days of instillation of the original model by increasing the concentration of BAC from 0.1% to 0.2%. MATERIALS AND METHODS: An experimental, prospective, and randomized study was performed on 10 male New Zealand white rabbits, divided into two groups, considering both eyes: 5 rabbits as control (n = 10) and 5 rabbits with 0.2% BAC treatment (n = 10). Saline solution (control) and 0.2% BAC were instilled for 5 consecutive days, twice daily. Tear secretion with and without anesthesia, tear breakup time, tear osmolarity, corneal staining, conjunctival hyperemia, density of goblet cells, height of mucin cloud, and transcript levels of IL-6 were measured before and after the treatment. RESULTS: After the instillation of 0.2% BAC for 5 consecutive days, there was a significant increase in tear secretion without anesthesia (P < 0.001), corneal staining (P < 0.001), conjunctival hyperemia (P < 0.001), and levels of IL-6 mRNA (P=0.005) compared to the control group. Conversely, there was a decrease in tear secretion with anesthesia (P < 0.001), tear breakup time (P=0.007), tear osmolarity (P < 0.001), density of goblet cells (P < 0.001), and height of mucin cloud (P < 0.001). CONCLUSIONS: The topical instillation of 0.2% BAC for 5 consecutive days, twice daily, was a proper procedure to induce a rabbit dry eye model, reducing the number of days of instillation compared to the original model (14 days).

Effect of Melatonin and Its Analogs on Tear Secretion.

Melatonin has been shown to enhance tear secretion associated with dinucleotide diadenosine tetraphosphate. This study investigated the isolated action of melatonin and its analogs, agomelatine, N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl) ethanamine (IIK7), and 5-methoxycarbonylamino-N-cetyltryptamine (5-MCA-NAT) (10 µl at 100 µM), on tear secretion when applied topically in the rabbit cornea and its relationship with the melatonin MT1, MT2, and MT3/quinone reductase QR2 receptors. The results showed a significant increase in tear secretion, with a maximal effect at 60 minutes for the agonists (138.9% ± 6.5%, 128.9% ± 6.4%, and 120.0% ± 5.2%, respectively; P < 0.05; 100% control) but not for melatonin (101.6% ± 7.9%; P > 0.05). Agonist action was tested combined with the antagonists DH97 (MT2 selective), prazosin (MT3/QR2 inhibitor), and luzindole (nonselective MT membrane receptor) (10 µl at 100 µM). DH97 reversed the effect of agomelatine, IIK7, and 5-MCA-NAT up to 30.85% ± 7.6%,108% ± 7.2%, and 87.01% ± 7.6%, respectively (P < 0.05; 100% control). Luzindole antagonized agomelatine and 5-MCA-NAT up to 67.35% ± 7.6% and 92.12% ± 8%, respectively (P < 0.05). Prazosin only reversed 5-MCA-NAT action up to 84.2% ± 7.7% (P < 0.05). These results suggest different pathways for the agonists to act through MT membrane receptors. Therefore, agomelatine, IIK7, and 5-MCA-NAT act through MT membrane receptors as secretagogues of tear secretion, and these analogs could be considered excellent therapeutic candidates for dry eye treatment. SIGNIFICANCE STATEMENT: Currently, dry eye with aqueous deficit is treated by adding artificial tears palliatively. This study shows that topical installation of three melatonin analogs (agomelatine, IIK7, and 5-MCA-NAT), but not melatonin, in therapeutic doses in the rabbit cornea significantly increases tear production, acting through different melatonin membrane receptor subtypes. Therefore, this study suggests that melatoninergic compounds could be considered excellent therapeutic candidates for dry eye treatment and ocular surface diseases occurring with a reduction in tear production.

The role and therapeutic potential of melatonin in age-related ocular diseases.

The eye is continuously exposed to solar UV radiation and pollutants, making it prone to oxidative attacks. In fact, oxidative damage is a major cause of age-related ocular diseases including cataract, glaucoma, age-related macular degeneration, and diabetic retinopathy. As the nature of lens cells, trabecular meshwork cells, retinal ganglion cells, retinal pigment epithelial cells, and photoreceptors is postmitotic, autophagy plays a critical role in their cellular homeostasis. In age-related ocular diseases, this process is impaired, and thus, oxidative damage becomes irreversible. Other conditions such as low-grade chronic inflammation and angiogenesis also contribute to the development of retinal diseases (glaucoma, age-related macular degeneration and diabetic retinopathy). As melatonin is known to have remarkable qualities such as antioxidant/antinitridergic, mitochondrial protector, autophagy modulator, anti-inflammatory, and anti-angiogenic, it can represent a powerful tool to counteract all these diseases. The present review analyzes the role and therapeutic potential of melatonin in age-related ocular diseases, focusing on nitro-oxidative stress, autophagy, inflammation, and angiogenesis mechanisms.

Low expression of CD39 and CD73 genes in centenarians compared with octogenarians.

Ageing involves a progressive decline of the body's regulatory systems including immune system. Adenosine regulates immune function by interaction with its receptors, mainly adenosine A2A receptor, present on the surface of immune cells. Furthermore, cellular response to this nucleoside is highly dependent on its extracellular concentration that is regulated by ecto-enzymes such as CD39 and CD73. Therefore, the aim of this study was to investigate the effect of age on adenosine A2A receptor, CD39 and CD73 gene expression. Changes in mRNA were measured by quantitative PCR from peripheral blood of young, middle-aged and older adults as well as centenarians. Centenarians showed a prominent decrease of CD39 and CD73 mRNA in comparison with older adults. Regarding to adenosine A2A receptor, we detected two subgroups of centenarians with high and low level of transcript. Additionally, adenosine A2A receptor mRNA level of centenarians, did not correlate with their cognitive impairment. In summary, our pilot study suggests that unlike of adenosine A2A receptor, the level of CD39 as well as CD73 mRNA could be a hallmark of successful human ageing.

Understanding the Presence and Roles of Ap4A (Diadenosine Tetraphosphate) in the Eye.

Diadenosine tetraphosphate abbreviated Ap4A is a naturally occurring dinucleotide, which is present in most of the ocular fluids. Due to its intrinsic resistance to enzyme degradation compared to mononucleotides, this molecule can exhibit profound actions on ocular tissues, including the ocular surface, ciliary body, trabecular meshwork, and probably the retina. The actions of Ap4A are mostly carried out by P2Y2 receptors, but the participation of P2X2 and P2Y6 in processes such as the regulation of intraocular pressure (IOP), together with the P2Y2, is pivotal. Beyond the physiological role, this dinucleotide can present on the ocular surface keeping a right production of tear secretion or regulating IOP. It is important to note that exogenous application of Ap4A to cells or animal models can significantly modify pathophysiological conditions and thus is an attractive therapeutic molecule. The ocular location where Ap4A actions have not been fully elucidated is in the retina. Although some analogues show interesting actions on pathological situations such as retinal detachment, little is known about the real effect of this dinucleotide, this being one of the challenges that require pursuing in the near future.

The role of dinucleoside polyphosphates on the ocular surface and other eye structures.

Dinucleoside polyphosphates comprises a group of dinucleotides formed by two nucleosides linked by a variable number of phosphates, abbreviated NpnN (where n represents the number of phosphates). These compounds are naturally occurring substances present in tears, aqueous humour and in the retina. As the consequence of their presence, these dinucleotides contribute to many ocular physiological processes. On the ocular surface, dinucleoside polyphosphates can stimulate tear secretion, mucin release from goblet cells and they help epithelial wound healing by accelerating cell migration rate. These dinucleotides can also stimulate the presence of proteins known to protect the ocular surface against microorganisms, such as lysozyme and lactoferrin. One of the latest discoveries is the ability of some dinucleotides to facilitate the paracellular way on the cornea, therefore allowing the delivery of compounds, such as antiglaucomatous ones, more easily within the eye. The compound Ap4A has been described being abnormally elevated in patient's tears suffering of dry eye, Sjogren syndrome, congenital aniridia, or after refractive surgery, suggesting this molecule as biomarker for dry eye condition. At the intraocular level, some diadenosine polyphosphates are abnormally elevated in glaucoma patients, and this can be related to the stimulation of a P2Y2 receptor that increases the chloride efflux and water movement in the ciliary epithelium. In the retina, the dinucleotide dCp4U, has been proven to be useful to help in the recovery of retinal detachments. Altogether, dinucleoside polyphosphates are a group of compounds which present relevant physiological actions but which also can perform promising therapeutic benefits.

Melatonin receptors trigger cAMP production and inhibit chloride movements in nonpigmented ciliary epithelial cells.

Melatonin and its analog 5-MCA-NAT (5-methylcarboxyamino-N-acetyl tryptamine) are active compounds reducing intraocular pressure (IOP). This action is mediated through MT2 and the putative MT3 melatonin receptor, producing a transient reduction of IOP that lasts for a few hours and has not yet been characterized. The use of melatonin and its analog are causing a decrease in chloride efflux from rabbit nonpigmented epithelial cells (NPE), possibly explaining the decrease in IOP. Melatonin and 5-MCA-NAT inhibited rabbit NPE chloride release in a concentration-dependent manner, whereas the pD2 values were between 4.5 ± 1.2 and 4.4 ± 1.0, respectively. Melatonin hypotensive action was enhanced by the presence of MT2 antagonists, such as DH97 (N-pentanoyl-2-benzyltryptamine) and 4-P-P-DOT (4-phenyl-2-propionamidotetralin) and by the nonselective melatonin receptor antagonist luzindole. Prazosin (1.5 µM) partially reverses the melatonin action by acting as a selective MT3 antagonist. However, at 15 nM it acts as an α-adrenergic receptor antagonist, enhancing the melatonin effect. Regarding the intracellular pathways triggered by melatonin receptors, neither phospholipase C/protein kinase C pathway nor the canonical reduction of intracellular cAMP was responsible for melatonin or 5-MCA-NAT actions. On the contrary, the application of these substances produced a concentration-dependent increase of cAMP, with pD2 values of 4.6 ± 0.2 and 4.9 ± 0.7 for melatonin and 5-MCA-NAT, respectively. In summary, melatonin reduces the release of chloride concomitantly to cAMP generation. The reduction of Cl(-) secretion accounts for a decrease in the water outflow and therefore a decrease in aqueous humor production. This could be one of the main mechanisms responsible for the reduction of IOP after application of melatonin and 5-MCA-NAT.

Signs and Symptoms of Dry Eye in Keratoconus Patients: A Pilot Study.

PURPOSE: To compare signs and symptoms of dry eye in keratoconus (KC) patients versus healthy subjects. METHODS: A total of 15 KC patients (KC group, n = 15 eyes) and 16 healthy subjects (control group, 16 eyes) were enrolled in this study. The Schirmer I test with no anesthetic, tear break-up time (TBUT), corneal staining characteristics, and ocular surface disease index (OSDI) scores were evaluated for both groups. Impression cytology, combined with/scanning laser confocal microscopy (LCM), was performed to evaluate goblet cell density, mucin cloud height (MCH), and goblet cell layer thickness (CLT). Finally, tear concentrations of di-adenosine tetraphosphate (Ap4A) were assessed. Results were statistically analyzed using Shapiro-Wilk and non-parametric Wilcoxon rank sum tests. Statistical significance was set at p < 0.05. RESULTS: KC patients had lower tear volumes and greater corneal staining than did healthy subjects (p < 0.05). OSDI scores were 44.96 ± 8.65 and 17.78 ± 6.50 for the KC and control groups, respectively (p < 0.05). We found no statistically significant differences in TBUT between groups. Impression cytology revealed lower goblet cell densities in KC group patients versus control group subjects (84.88 ± 32.98 and 128.88 ± 50.60 cells/mm,(2) respectively, p < 0.05). There was a statistically significant reduction in MCH and CLT in KC group patients compared with control group subjects. Ap4A tear concentrations were higher in KC group patients than in control group subjects (2.56 ± 1.10 and 0.15 ± 0.12 µM, respectively, p < 0.05). CONCLUSIONS: The parameters evaluated in this study indicate that KC patients suffer greater symptoms of dry eye and greater tear instability, primarily due to the decreased mucin production in their tears, than do healthy patients with no KC.

Effect of melatonin and analogues on corneal wound healing: involvement of Mt2 melatonin receptor.

PURPOSE: We have investigated the effect of melatonin and its analogues on rabbit corneal epithelial wound healing. METHODS: New Zealand rabbits were anaesthetised and wounds were made by placing Whatman paper discs soaked in n-heptanol on the cornea. Melatonin and analogues (all 10 nmol) were instilled. Wound diameter was measured every 2 hours by means of fluorescein application with a Topcon SL-8Z slit lamp. Melatonin antagonists (all 10 nmol) were applied 2 hours before the application of the n-heptanol-soaked disc and then every 6 hours together with melatonin. To confirm the presence of MT2 receptors in corneal epithelial cells immunohistochemistry, Western blot and RT-PCR assays in native tissue and in rabbit corneal epithelial cells were performed. The tear components were extracted then processed by HPLC to quantify melatonin in tears. RESULTS: Migration assays revealed that melatonin and particularly the treatment with the MT2 agonist IIK7, accelerated the rate of healing (p < 0.001). The application of the non-selective melatonin receptor antagonist luzindole and the MT2 antagonist DH97 (but not prazosin), prevented the effect of melatonin on wound healing (both p < 0.001). Immunohistochemistry, Western blot and RT-PCR assays showed the presence of MT2 melatonin receptor in corneal epithelial cells. In addition, we have identified melatonin in tears and determined its daily variations. CONCLUSIONS: These data suggest that MT2 receptors are implicated in the effect of melatonin on corneal wound healing regulating migration rate. This suggests the potential use of melatonin and its analogues to enhance epithelial wound healing in ocular surface disease.

An update on dry eye disease molecular treatment: advances in drug pipelines.

INTRODUCTION: Dry eye disease is a common disorder provoking changes in tear film and ocular surface. Untreated dry eye could cause ocular infections, corneal ulcer and blindness. Only a few drugs are authorized so far for the treatment of dry eye disease and the possibilities of evolution in this sector are immense. Consequently, a significant number of new potential solutions are under development or placed in the pharmaceutical pipeline, promising better results and lesser side effects. AREAS COVERED: In this article, the corresponding literature and recent Phase III clinical trial data and the corresponding literature, for dry eye disease treatment are reviewed, revealing the new strategic movements in drug pipelines. EXPERT OPINION: From the clinical trial results, the advancement in tear substitutes and secretagogues in addressing specific deficiencies of tear components even though not resolving the underlying conditions of the disease is evident. The vast majority of new compounds under development are anti-inflammatories, steroids, non-steroids and antibiotics; however, there are also some novel lubricating drops and mucin-tear secretagogues. A future aggressive therapy for dry eye, depending on the severity of the symptoms, would include combinations of soft steroids, anti-inflammatories, such as cyclosporine A, with the addition of the new polyvalent mucin and tear secretagogues.

Melatonin and its analog 5-methoxycarbonylamino-N-acetyltryptamine potentiate adrenergic receptor-mediated ocular hypotensive effects in rabbits: significance for combination therapy in glaucoma.

Melatonin is currently considered a promising drug for glaucoma treatment because of its ocular hypotensive and neuroprotective effects. We have investigated the effect of melatonin and its analog 5-methoxycarbonylamino-N-acetyltryptamine, 5-MCA-NAT, on ß2/α(2A)-adrenergic receptor mRNA as well as protein expression in cultured rabbit nonpigmented ciliary epithelial cells. Quantitative polymerase chain reaction and immunocytochemical assays revealed a significant ß2-adrenergic receptor downregulation as well as α(2A)-adrenergic receptor up-regulation of treated cells (P < 0.001, maximal significant effect). In addition, we have studied the effect of these drugs upon the ocular hypotensive action of a nonselective ß-adrenergic receptor (timolol) and a selective α2-adrenergic receptor agonist (brimonidine) in normotensive rabbits. Intraocular pressure (IOP) experiments showed that the administration of timolol in rabbits pretreated with melatonin or 5-MCA-NAT evoked an additional IOP reduction of 14.02% ± 5.8% or 16.75% ± 5.48% (P < 0.01) in comparison with rabbits treated with timolol alone for 24 hours. Concerning brimonidine hypotensive action, an additional IOP reduction of 29.26% ± 5.21% or 39.07% ± 5.81% (P < 0.001) was observed in rabbits pretreated with melatonin or 5-MCA-NAT when compared with animals treated with brimonidine alone for 24 hours. Additionally, a sustained potentiating effect of a single dose of 5-MCA-NAT was seen in rabbits treated with brimonidine once daily for up 4 days (extra IOP decrease of 15.57% ± 5.15%, P < 0.05, compared with brimonidine alone). These data confirm the indirect action of melatoninergic compounds on adrenergic receptors and their remarkable effect upon the ocular hypotensive action mainly of α2-adrenergic receptor agonists but also of ß-adrenergic antagonists.

Ocular disorders and the utility of animal models in the discovery of melatoninergic drugs with therapeutic potential.

INTRODUCTION: The pineal indole-derived hormone melatonin is a modulator of circadian and seasonal rhythms with an important role in ocular health and disease. This could be due to specific melatonin receptors that have been identified in structures such as cornea, lens, ciliary body, retina, choroid and sclera. In addition, a local synthesis of melatonin occurs in several of these ocular tissues. AREAS COVERED: The authors review existing literature on the most common animal models where ocular melatonin actions have been tested. The therapeutic potential of melatonin in diabetic keratopathy and retinopathy, keratitis, cataracts, glaucoma, uveitis, age-related macular degeneration and retinitis pigmentosa is discussed. Furthermore, the authors comment on the usefulness of different animal models for the development of melatoninergic drugs with therapeutic potential. EXPERT OPINION: The use of animals for the study of ocular diseases and the potentiality of melatonin and its analogs, as future therapeutic drugs, should be performed on the basis of a rationale study. It is important to note that melatonin receptors seem to be widespread all over the eye. This strongly suggests that, in order to modify the physiology and biochemistry of malfunctioning ocular tissue, the melatonin receptors which are present in that tissue must be first identified. Second there is the need to confirm that those receptors targeted perform the desirable responses, and as a third measure, to use selective agonists (or antagonists) instead of melatonin. However, although some animals mimic ocular pathologies relatively well, and these can be used in melatonin studies, there is still a long way to go till some of the results obtained in animal models could be used for human therapy.

Recent patents and developments in glaucoma biomarkers.

Glaucoma is an eye condition mainly developed from an excessive intraocular pressure. The condition tends to be inherited and may not show up until later in life. The increased pressure, can damage the optic nerve, provoking loss of vision. Without treatment, glaucoma can cause blindness within a few years; consequently glaucoma has to be diagnosed before long-term visual loss occurs. If it is diagnosed and treated early, the disease can be controlled. Usually, the patient does not notice any early symptoms or pain from this increased pressure, so the early diagnosis is problematic. Over half of the patients with glaucoma are unaware they have this blinding disease and by the time they are diagnosed, they already have irreversibly lost approximately 30-50% of their retinal ganglion cells. Glaucoma diagnosis is currently based on specific signs of the disease, characteristic optic nerve head changes and visual field loss. Thus, improved methods for early diagnosis of glaucoma are needed. Molecular genetics are valuable for the understanding the pathophysiology and cure of glaucoma, but still are not widely used for its diagnosis. Genetic studies on glaucoma have revealed many genes and chromosomal loci associated to glaucoma. Consequently, a better understanding of the molecular mechanisms underlying glaucoma is required to obtain early diagnosis and avoid potential disease progression. In this article, we revise the patents and the corresponding literature on the latest developments and approaches in glaucoma diagnosis, using mainly molecular genetics.