Role of instrumental factors in Meibomian gland contrast assessment.

PURPOSE: Meibomian gland contrast has been suggested as a potential biomarker in Meibomian gland dysfunction. This study analysed the instrumental factors related to contrast. The objectives were to determine whether the mathematical equations used to compute gland contrast (e.g., Michelson or Yeh and Lin), impact the ability to identify abnormal individuals, to ascertain whether contrast between the gland and the background could be an effective biomarker and to assess whether using contrast-enhancement on the gland image improves its diagnostic efficacy. METHODS: A total of 240 meibography images from 40 participants (20 controls and 20 having Meibomian gland dysfunction or blepharitis), were included. The Oculus Keratograph 5M was used to capture images from the upper and lower eyelids of each eye. The contrast of unprocessed images and those pre-processed with contrast-enhancement algorithms were analysed. Contrast was measured on the eight central glands. Two equations for contrast computation were used, and the contrast both between glands and within a gland were calculated. RESULTS: Significant differences were found between the groups for inter-gland area in the upper (p = 0.01) and lower eyelids (p = 0.001) for contrast measured with the Michelson formula. Similar effects were observed when using the Yeh and Lin method in the upper (p = 0.01) and lower eyelids (p = 0.04). These results were obtained for images enhanced with the Keratograph 5M algorithm. CONCLUSIONS: Meibomian gland contrast is a useful biomarker of disease related to the Meibomian glands. Contrast measurement should be determined using contrast-enhanced images in the inter-gland area. However, the method used to compute contrast did not influence the results.

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.

Importance of Lid Hygiene Before Ocular Surgery: Qualitative and Quantitative Analysis of Eyelid and Conjunctiva Microbiota.

OBJECTIVE: To evaluate the efficacy of a cleansing eyelid wipe in reducing the microbiota present on the ocular surface before cataract surgery. METHODS: A single-center, prospective, single-blind phase IV study was conducted at the University Complutense of Madrid. Forty-five adult patients who were scheduled for ocular surgery after treatment with commercially available eyelid wipes were consecutively enrolled. The study lasted 5 days and the patients were examined at day 0 (D0), day 3 (D3), and day 5 (D5). They received instructions to apply the eyelid wipe only to the eye subject to surgery, using the other eye as a control with no treatment. Lid and conjunctival swabs were taken on each day and microbes identified. Ocular surface microbiota was estimated by measuring the area of the agar plate occupied by the grown colonies with respect to the total available area. RESULTS: Measurements at D3 and D5 showed a percent reduction of 58% and 63%, respectively, in the microbial load on the eyelid in the treated eyes (P=0.0011). There was also a reduction, although nonsignificant, in the microbiota of the conjunctiva of 72% and 69% on D3 and D5, respectively. CONCLUSIONS: The degree of microbiota reduction was comparable with that obtained after topical application of antibiotics in other studies. The results suggest the use of these eyelid wipes as a complementary prophylactic method before any ocular surgery.

A Comparative Study of Two Imaging Techniques of Meibomian Glands.

In the present study, two different meibographers, Oculus Keratograph 5M (K5M) that uses 840 nm infrared light and the Visible Light Non-Contact Meibographer (VLNCM) that uses 610 nm visible light have been used to obtain meibography images from normal and Meibomian Gland Dysfunction (MGD) population. The main objective has been to validate and demonstrate that the use of visible light is useful for observation and quantification of MG in clinical practice. Twenty participants were enrolled in this prospective study. The upper eyelids of one randomly chosen eye were used to obtain results. Forty images were captured and analysed. Three specialized observers were recruited to grade images using Pult and Riede Pult 5-degree scale, in two different sessions. Intra-observer agreement between sessions for both devices was shown. Inter-observer variability analysis showed discrepancy between meiboscores obtained from observers with K5M (p-value < 0.05), except for session 2 in the pathology group, while no statistical difference was found with VLNCM. Repeatability analysis found no statistically significant differences between sessions. Correlation between meibographers showed no statistically significant difference and a moderate correlation coefficient between meiboscores graded with the two devices. The current study suggests that VLNCM can allow MG to be properly visualized and classified in the upper eyelids.

Improving ocular surface comfort in contact lens wearers.

PURPOSE: Contact lens discomfort (CLD) is a major concern that can lead to the decreased or abandoned use of contact lenses. Contact lens users with dry eye disease are more likely to present with CLD. This study was conducted to evaluate the efficacy of a bioprotective preservative free, hypotonic, 0.15% hyaluronic acid (HA)-3% Trehalose artificial tear in managing dry eye symptoms in contact lens wearers. METHODS: A prospective, single-arm, observational pilot study to evaluate the effectiveness of treatment with HA-Trehalose artificial tears in contact lens wearers (N = 33) aged 18-45 years with symptoms of ocular discomfort. Participants used a preservative-free, hypotonic HA-Trehalose artificial tear (1 drop/4 times per day) for 84 days. Participants were assessed using Visual Analogue Scale (VAS) for dry eye symptoms (pain, photophobia, dry eye sensation, blurry vision, foreign body sensation, itching, tingling/burning, and sticky eye feeling), Ocular Surface Disease Index (OSDI), Contact Lens Dry Eye questionnaire (CLDEQ-8), Berkley Dry Eye Flow-Chart (DEFC) on Day 0 and Day 84 and tear break-up time (TBUT), ocular surface staining with fluorescein and lissamine green, tear meniscus evaluation, and visual acuity on Day 0, 35, and 84. RESULTS: All VAS symptoms (except tingling/burning and sticky eye feeling), OSDI, CLEDQ-8, and DEFC showed statistically significant (p < 0.05) improvement from baseline (Day 0) to Day 84. Similarly, corneal (fluorescein) and conjunctival (lissamine green) quality improved during the study (p < 0.05 at Day 84 versus baseline). Tear break-up time (TBUT), conjunctival (lissamine green) staining, and tear meniscus decreased but the changes were not statistically significant. Visual acuity did not change during the study. There were no ocular or systemic adverse events. CONCLUSIONS: This study showed that the instillation of a preservative-free, hypotonic, HA-Trehalose artificial tear in contact lenses wearers with dry eye syndrome significantly improved symptoms and reduced associated signs such as corneal and conjunctival staining.

Design of novel melatonin analogs for the reduction of intraocular pressure in normotensive rabbits.

Melatonin, the MT(2) melatonin receptor agonist IIK7 [N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl)ethanamine], and the putative MT(3) melatonin receptor agonist 5-MCA-NAT [5-methoxycarbonylamino-N-acetyltryptamine] have previously been shown to reduce intraocular pressure (IOP) in ocular normotensive rabbits. To gain a better understanding of the structure-activity relationship of compounds that activate MT(2) and MT(3) receptors mediating reductions in IOP, novel melatonin analogs with rationally varied substitutions were synthesized and tested for their effects on IOP in ocular normotensive rabbits (n = 160). All synthesized melatonin analogs reduced IOP. The best-effect lowering IOP was obtained with the analogs INS48848 [methyl-1-methylene-2,3,4,9-tetrahydro-1H-carbazol-6-ylcarbamate], INS48862 [methyl-2-bromo-3-(2-ethanamidoethyl)-1H-indol-5-ylcarbamate], and INS48852 [(E)-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-3-phenylprop-2-enamide]. These compounds produced dose-dependent decreases in IOP that were maximal at 0.1 mM (total dose of 0.259 µg for INS48848, 0.354 µg for INS48862, and 0.320 µg for INS48852) and 1 mM (total dose of 2.59 µg for INS48848, 3.54 µg for INS48862, and 3.20 µg for INS48852), with maximal reductions of 36.0 ± 4.0, 24.0 ± 1.5, and 30.0 ± 1.5% for INS48848, INS48862, and INS48852, respectively. Studies using melatonin receptor antagonists (luzindole, prazosin, and DH97 [N-pentanoyl-2-benzyltryptamine]) indicated that INS48862 and INS48852 activate preferentially a MT(2) melatonin receptor and suggest that INS48848 may act mainly via a MT(3) receptor. The most effective compounds were also well tolerated in a battery of standard ocular surface irritation studies. The implication of these findings to the design of novel drugs to treat ocular hypertension is discussed.

Comfort and vision scores at insertion and removal during 1 month of wear of Paragon CRT for corneal reshaping.

PURPOSE: The goal of this study was to evaluate the pattern of initial adaptation of neophytes to corneal refractive therapy (CRT) for overnight corneal reshaping in terms of comfort and subjective visual performance at lens insertion at night and lens removal in the morning. METHODS: Twenty-two young healthy subjects were enrolled in this study. All of them had been trialed to assess adaptation to conventional alignment-fit rigid gas permeable lenses and were only enrolled in this study after a 2-week wash-out period. Visual analog scales for subjective comfort and vision were recorded on a form given to the patient on days 1, 2, 3, 5, 7, 14, 21, and 28. Additionally, the patient attended the clinic on days 1, 7, 15, and 30 after fitting, for follow-up. RESULTS: Successful adaptation was obtained in 21 of the 22 initially enrolled individuals. The average overnight wearing time remained constant during the study at 8 hrs per day. Overall comfort rates increased significantly up to values of 8.02 and 9.12 out of 10 at insertion and removal, respectively (P<0.001). Subjective vision scores also increased significantly at the end of the 1-month study period (P<0.001). CONCLUSIONS: Adaptation to CRT is rapid in terms of subjective comfort and vision. Comfort significantly increases by day 5, whereas subjective vision in the morning reaches its maximum by days 15 to 21 and at the end of the day by days 10 to 15. These results are of interest to clinicians to provide evidence-based information to their patients about the expected time to adapt to CRT in terms of self-reported comfort and vision.

Nucleotides in ocular secretions: their role in ocular physiology.

The eye is the sense organ that permits the detection of light owing to the existence of a sophisticated neuronal array, called the retina, which is responsive to photons. The correct functioning of this complex system requires the coordination of several intraocular structures that ultimately permit the perfect focusing of images on the neural retina. Light has to pass through different media: the tear, the cornea, aqueous humour, lens, and vitreous humour before it reaches the retina. Moreover, the composition and structure of some of these media can change due to several physiological mechanisms. Nucleotides are active components of the humours bathing relevant ocular structures. The tear contains nucleotides and dinucleotides that control the process of tearing, wound healing and protects of superficial infections. In the inner eye, the aqueous humour also presents a collection of mono and dinucleotides that affect pupil contraction, aqueous humour production and accommodation. Behind the lens and between this structure and the retina the vitreous humour can modify the physiology of the retinal cells, mostly the ganglion cells. By investigating the actions of nucleotides and dinucleotide present in the ocular humours we will be able not only to understand the functioning of the ocular structures but also to develop new pharmacological therapies for pathologies such as dry eye, glaucoma or retinal detachment.

Adenine nucleotide effect on intraocular pressure: Involvement of the parasympathetic nervous system.

Nucleotides are present in the aqueous humor possibly exerting physiological effects on intraocular pressure (IOP). To determine the effect of nucleotides such as ATP and its related derivatives on IOP, New Zealand white rabbits were used. IOP was measured in rabbits treated topically either with saline (control) or with a single dose (10 microg/microL) of adenine nucleotides (ATP, 2-meS-ATP, ATP-gamma-S, alpha,beta-meADP, alpha,beta-meATP and beta,gamma-meATP). Those nucleotides reducing IOP (alpha,beta-meATP and beta,gamma-meATP) were then tested in concentrations ranging from 1 to 100 microg/microL to obtain the IC(50) value. Several antagonists for the P2 and adenosine A1 receptors (all at 10 microg/microL) were assayed 30 min before the application of the hypotensive nucleotide beta,gamma-meATP. To see whether the nucleotide was acting directly on the structures involved in aqueous humor dynamics or on the autonomic nerves controlling IOP, animal denervation and sympathetic (yohimbine and ICI-118,551 at 10 microg/microL) and parasympathetic (atropine and hexametonium at 10 microg/microL) receptors' antagonists were used 30 min before the instillation of beta,gamma-meATP. alpha,beta-meATP and beta,gamma-meATP decreased IOP to 60% of control value (basal IOP=23.2+/-1.3 mmHg), with IC(50) of 1.59+/-0.21 microg/microLand 0.56+/-0.62 microg/microL, which corresponds to 3mM and 1mM respectively. Denervation completely abolished the effect of beta,gamma-meATP. Sympathetic antagonists did not modify the hypotensive effect of beta,gamma-meATP, but parasympathetic antagonists were able to abolish it. Among the series of adenine nucleotide tested, alpha,beta-meATP and beta,gamma-meATP presented hypotensive actions on IOP. beta,gamma-meATP seems to stimulate cholinergic terminals being its final effect the IOP reduction. Therefore, these two nucleotides are interesting pharmacological tools for those pathologies related with high intraocular pressure.

Ophthalmic formulations of the intraocular hypotensive melatonin agent 5-MCA-NAT.

Melatonin is a hormone responsible for the regulation of circadian and seasonal rhythms. This hormone is synthesised in many tissues in the body including the eye, where it regulates important processes. During the recent years, the role of melatonin in the control of IOP has been investigated and it has been demonstrated that melatonin receptors are present and involved in the dynamics of the aqueous humour. 5-Methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) is a selective MT3 melatonin receptor agonist. Topical application of this product produces a clear reduction in intraocular pressure (IOP) in New Zealand white rabbits and in glaucomatous monkeys. In this work, the potent ocular hypotensive 5-MCA-NAT has been dissolved in excipients used in currently marketed drug formulations. Until now, this melatonin analogue had been dissolved in either DMSO or ethanol neither of which is suitable for ocular topical application in humans. Solubility assays in the different solvents were performed by the observation of the presence of drug crystals under optical microscopy. 5-MCA-NAT was completely dissolved in propylene glycol (PG) and polyethylene glycol 300 (PEG 300) within 24h. Ophthalmic formulations were prepared from different ratios of PG:PBS and the commercialized Systane product. Quantification of 5-MCA-NAT in the vehicles was assessed by HPLC. In vitro cytotoxicity of the formulations was evaluated by the MTT method and in vivo tolerance of 5-MCA-NAT in the solvents was analyzed by biomicroscopy and specular microscopy. Systane and proportions of PG:PBS up to 10% of PG did not show cytotoxicity in human corneal limbal epithelial cells (HCLE). In vivo experiments showed that the higher the ocular tolerance, the less amount of PG present. The ocular hypotensive effect of 5-MCA-NAT dissolved in the new formulations was checked measuring IOP for 8h after instillation of the substance. The best effect lowering IOP was obtained with 5-MCA-NAT dissolved in PG and diluted with PBS (PG 1.43%) in which 5-MCA-NAT produced a reduction of 28.11+/-2.0% and the effect lasted about 7h. In conclusion, new formulations accepted for ocular topical treatments different from DMSO or ethanol were capable of dissolving the melatonin analogue 5-MCA-NAT, preserving its ocular hypotensive ability. Therefore, the use of 5-MCA-NAT may be possible in the treatment of ocular hypertension and glaucoma.

Dinucleoside polyphosphates in the eye: from physiology to therapeutics.

Diadenosine polyphosphates are a family of dinucleotides with emerging biochemical, physiological, pharmacological and therapeutic properties in the eye and other tissues. These compounds are formed by two adenosine moieties linked by their ribose 5'-ends to a variable number of phosphates. Diadenosine polyphosphates are present as active components of ocular secretions such as tears and aqueous humour and they can activate P2 purinergic receptors present on the ocular surface, anterior segment and retina. Both metabotropic and ionotropic actions mediated by P2Y and P2X receptors, respectively are responsible for the control of processes such as induction of tear secretion, lysozyme production or acceleration of corneal wound healing. Inside the eye the dinucleotide Ap(4)A can reduce intraocular pressure by acting on P2Y(1) receptors present in trabecular meshwork cells and on P2X(2) receptors present on the cholinergic terminals located in the ciliary muscle. In the retina, derivatives of diadenosine polyphosphates can improve the re-absorption of fluids in retinal detachment. Altogether, diadenosine polyphosphates are not only dinucleotides with roles in the physiology of the eye but it is also possible that their properties may serve to help in the treatment of some ocular pathologies.

Therapeutic targets in dry eye syndrome.

Dry eye is a multifactorial disease of the tears and the ocular surface that manifests with a wide variety of signs and symptoms. It is prevalent in about 33% of the population worldwide. Due to the importance of the pathology, new tests, drugs and technologies have been developed to assist the diagnosis, management and follow-up of the disease. Current available therapies try to alleviate symptoms and to reduce signs in order to restore the ocular surface. Depending on the etiology of the pathology it is possible to use lubricants, secretagogues, biological tear substitutes or antiinflammatory drugs, either independently or combined. Nowadays, the therapies under clinical trial are devoted to stimulating tear components (e.g., diquafosol, a P2Y receptor agonist), or mucin secretion (e.g., rebamipide, an amino acid analogue of quinolinone). Others include gefarnate, a water-insoluble terpene fatty acid that contributes to restoring mucins on the ocular surface, or cevimeline, an oral cholinergic agonist that reduces the symptoms associated with dry eye. Other potential compounds described in patents are in a lower phase of drug development. These compounds come from different families of therapies, and among others, can be found in the form of steroidal and nonsteroidal antiinflammatory agents, vitamins A and D, neurotransmitters and neuropeptides.

Hypotensive effect of UDP on intraocular pressure in rabbits.

Nucleotides can modify intraocular pressure (IOP). We have tested the ability of uridine-5'-diphosphate, UDP, for modulating IOP in New Zealand white rabbits. Uridine 5' diphosphate, UDP, reduced IOP by 82.9+/-2.6% compared to control. Dose-response analysis demonstrated a concentration dependent pattern which presented a pD(2) value of 7.57+/-1.45, equivalent to an EC(50) of 26.91 nM. Of all the tested P2 receptor antagonists, suramin, pyridoxalphosphate-6-azophenyl-2, 4-disulfonic acid (PPADS) and Reactive Blue 2 (RB-2), only the last two were able to reverse the action triggered by UDP. Altogether, UDP acting probably on P2Y(6) receptors present on the ciliary processes, can reduce intraocular pressure, indicating that this substance may be used for the treatment of ocular hypertension and glaucoma.

Ocular mucin visualization by confocal laser scanning microscopy.

PURPOSE: To describe a new method of visualizing human conjunctiva goblet cell mucin secretion by using a combination of impression cytology and laser scanning microscopy. METHODS: By assembling a Z-stack of confocal microscopy images taken from human impression cytology samples, we obtained 3-dimensional information about the release and spread of goblet cell secretions above the conjunctival surface. After reconstruction and rendering of these images, analysis of the shape and spreading characteristics of the mucins permitted definition of the following parameters related to goblet cell secretion: mucin cloud height as the height of the top of the cloudlike mucin structure visible above the goblet cell opening and spread mucin thickness, which is the thickness of the mucin layer distributed over the surface of the conjunctiva. RESULTS: Several impression cytology samples of control and muco-deficient patients have been analyzed through the confocal laser scanning technique, and significant differences between these groups were found. Mucin cloud height and spread mucin thickness values for controls were 8.81 +/- 4.00 and 2.77 +/- 1.00 microm, respectively (n = 25). These values decreased by approximately 70% and 40%, respectively, for moderately mucodeficient subjects and by 84% and 48% for those with severe mucodeficiency. Classifying those individuals having mucin-related pathology may thus be possible on the basis of application of these techniques. CONCLUSIONS: In summary, we present a method of objectively identifying those individuals with problems associated with either a lack of mucins or a reduction in the distribution of these proteins over the ocular surface.

Hypotensive effect of profilin on rabbit intraocular pressure.

Ocular hypertension is a negative process that occurs within the eye and is the main risk factor to develop glaucoma, a progressive loss of vision due to degeneration of retinal ganglion cells. The protein transduction technique allows a cargo to cross biological membranes. Using this technique we have previously shown that a membrane permeable version of profilin I (PTD4-profilin) increased aqueous humour outflow facility. Here we have investigated if a topical application of PTD4-profilin was able to modify intraocular pressure in rabbits. 10 microM PTD4-profilin (10 microL), reduced intraocular pressure by 20% compared to the control vehicle, this value being in the range of other commercial drugs, which produced intraocular pressure reductions between 18 and 35%. The mean-time effect for PTD4-profilin was 6.8 h and was also in the same range as commercial products that provided values between 4.3 and 5.5 h. According to the results presented here we propose PTD4-profilin as a new approach for the treatment of ocular hypertension and PTD4 as a new strategy to facilitate the penetration of molecules into the eye.

Requirement of intact sympathetic transmission for the ocular hypotensive effects of melatonin and 5-MCA-NAT.

Melatonin and its analogue, 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), potently reduce intraocular pressure, and may be good candidates for the treatment of ocular hypertension and glaucoma. After chemical sympathectomy by reserpine or 6-hydroxydopamine, the hypotensive effects of melatonin and 5-MCA-NAT are severely inhibited. This indicates that the sympathetic nervous system is involved in the production and drainage of aqueous humour by the ciliary body and trabecular meshwork, and that it mediates the effects of melatonin and its analogue, 5-MCA-NAT.

Dual roles of diadenosine polyphosphates in corneal epithelial cell migration.

PURPOSE: To investigate the influence of diadenosine polyphosphates on the rate of corneal epithelial cell migration. METHODS: Primary corneal epithelial cell cultures were obtained from New Zealand White rabbits. Immunocytochemical experiments were performed by fixing the cells with 4% paraformaldehyde (PFA) and incubated with cytokeratin 3 primary antibody, which was subsequently incubated with a secondary IgG mouse labeled with FITC, and the cells were observed under confocal microscopy. Migration studies were performed by taking confluent monolayers that were wounded with a pipette tip and challenged with different di- and mononucleotides with or without P2 antagonist (n = 8 each treatment). For concentration-response analysis, compounds were tested in doses ranging from 10(-8) to 10(-3) M (n = 8). The stability of the dinucleotides was assayed by HPLC, with an isocratic method (n = 4). RESULTS: Cells under study were verified as corneal epithelial cells via the immunocytochemical analysis. Cell migration experiments showed that Ap4A, UTP, and ATP accelerated the rate of healing (5, 2.75, and 3 hours, respectively; P < 0.05; P < 0.001), whereas Ap3A, Ap5A, and UDP delayed it (6.5, 10, and 2 hours, respectively; P < 0.05). ADP did not modify the rate of migration. Antagonists demonstrated that Ap4A and Ap3A did activate different P2Y receptors mediating corneal wound-healing acceleration and delay. Concerning the possible degradation of the dinucleotides, it was almost impossible to detect any products resulting from their cleavage. CONCLUSIONS: Based on the pharmacological profile of all the compounds tested, the two main P2Y receptors that exist in these corneal cells are a P2Y(2) receptor accelerating the rate of healing and a P2Y6 receptor that delays this process.

Increased levels of diadenosine polyphosphates in dry eye.

PURPOSE: To analyze the levels of the diadenosine polyphosphates Ap4A and Ap5A in tears, in a set of control subjects and in groups of symptomatic and nonsymptomatic persons with dry eye. METHODS: Ninety-seven subjects participated in the study. The subjects were divided into five experimental groups: control subjects; symptomatic patients with normal tear secretion; symptomatic patients with low tear secretion; forced blink; and corneal mechanical stimulation provided by a gas esthesiometer. The Schirmer I test was used to measure and collect tear secretions from each subject. All samples were processed by high pressure liquid chromatography (HPLC) and their Ap4A and Ap5A levels determined. RESULTS: The levels of Ap4A and Ap5A in tears were greater in all symptomatic patients than in control subjects, especially in symptomatic subjects with low tear secretion. Within the symptomatic subjects with normal tear secretion, significant differences in concentrations of Ap4A and Ap5A were found between men and women. In the forced blink experiments, concentrations of the Ap4A and Ap5A rose with increasing blink frequency. When the cornea was mechanically stimulated, the levels of Ap4A and Ap5A rose significantly during both moderate and high-flow rate tests. CONCLUSIONS: The increased levels of Ap4A and Ap5A in tears of patients with dry eye allow these dinucleotides to be used as objective biomarkers in dry eye conditions.

Melatonin potentiates tear secretion induced by diadenosine tetraphosphate in the rabbit.

Diadenosine tetraphosphate (Ap(4)A, 0.03 nmol) applied topically to the cornea of New Zealand white rabbits, evoked an increase in tear secretion of 9.7 +/- 2.60% (N=7). Melatonin (1 nmol) had no significant effect. Application of Ap(4)A in combination with melatonin, evoked a significantly greater increase in tear secretion of 34.2 +/- 5.8% (N=11). This potentiating effect of melatonin was blocked by pretreating the cornea with a topical application of the melatonin receptor antagonist, luzindole (240 nmol). Melatonin combined with Ap(4)A may be useful for treating dry eye conditions.

Diadenosine polyphosphates in the tears of aniridia patients.

PURPOSE: To quantify diadenosine polyphosphate levels in tears of congenital aniridia patients to estimate the ocular surface changes associated with congenital aniridia compared to normal individuals. METHODS: Fifteen patients diagnosed with congenital aniridia and a control group of forty volunteers were studied. Tears were collected to quantify the levels of diadenosine polyphosphates Ap4 A and Ap5 A by high-performance liquid chromatography (H.P.L.C). Break-up time (BUT), corneal staining, McMonnies questionnaire and the Schirmer I test were applied to both groups. RESULTS: Dinucleotides in congenital aniridia patients were higher than in control subjects. For the congenital aniridia group, under 15 years old, the values were 0.77 ± 0.01 µm and 0.17 ± 0.02 µm for Ap4 A and Ap5 A, respectively. The group aged from 15 to 40 years old provided concentrations of 4.37 ± 0.97 µm and 0.46 ± 0.05 µm for Ap4 A and Ap5 A, the group over 40 gave concentrations of 11.17 ± 5.53 µm and 0.68 ± 0.17 µm for Ap4 A and Ap5 A. Dinucleotide concentrations increased with age, being statistically significant different among the three age groups (p < 0.05). Congenital aniridia patients showed a normal tear secretion and no dry eye McMonnies scores, except for the group over 40 years old. BUT values decreased and corneal staining increased with age and correlated with the levels of diadenosine polyphosphates (p < 0.05). CONCLUSIONS: The levels of dinucleotides in tears increase in aniridia patients compared with healthy subjects, and they seem to be related with the progression of corneal disorders in aniridia patients, both of which increase with ageing.