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.

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.

Comparison of Intraocular Pressure before and after Laser In Situ Keratomileusis Refractive Surgery Measured with Perkins Tonometry, Noncontact Tonometry, and Transpalpebral Tonometry

Purpose. To compare the intraocular pressure (IOP) before and after Laser In Situ Keratomileusis (LASIK), measured by Diaton, Perkins, and noncontact air pulse tonometers. Methods. Fifty-seven patients with a mean age of 34.88 were scheduled for myopia LASIK treatment. Spherical equivalent refraction (SER), corneal curvature (K), and central corneal thickness (CCT) and superior corneal thickness (SCT) were obtained before and after LASIK surgery. IOP values before and after surgery were measured using Diaton, Perkins, and noncontact air pulse tonometers. Results. The IOP values before and after LASIK surgery using Perkins tonometer and air tonometers were statistically significant (). However, no significant differences were found () for IOP values measured with Diaton tonometer. CCT decreases significantly after surgery () but no statistical differences were found in SCT (). Correlations between pre- and postsurgery were found for all tonometers used, with and for the air pulse tonometer, and for Perkins, and and for Diaton. Conclusion. Transpalpebral tonometry may be useful for measuring postsurgery IOP after myopic LASIK ablation because this technique is not influenced by the treatment.

Corneal Re-epithelialization Stimulated by Diadenosine Polyphosphates Recruits RhoA/ROCK and ERK1/2 Pathways

Purpose. To investigate the role of ERK1/2 and RhoA/ROCK intracellular pathways in the modification of corneal re-epithelialization when stimulated by the diadenosine polyphosphates Ap4A and Ap3A. Methods. In wounded confluent SIRC (Statens Seruminstitut rabbit cornea) cell monolayers and in the presence or absence of Ap4A or Ap3A 100 μM, a battery of P2 receptor antagonists and inhibitors of tyrosin kinases, MAPK, and cytoskeleton pathways (AG1478 100 μM, U0126 100 μM, Y27632 100 nM, and (−)-blebbistatin 10 μM; n = 8 each) were assayed. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with Ap4A and Ap3A (100 μM), with or without suramin, RB-2, U0126, and Y27632. The intracellular distribution of pERK and ROCK-I was examined in the presence of Ap4A or Ap3A (100 μM) with U0126 and Y27632 (100 nM). Results. In the presence of Ap4A, U0126, Y27632, AG1478, and (−)-blebbistatin, reduced the migration rate compared to the effect of Ap4A alone (P < 0.0001, P < 0.001, P < 0.01, and P < 0.1 versus Ap4A, respectively). In the presence of Ap3A 100 μM, U0126 and Y27632 accelerated the migration rate when compared with the effect of Ap3A alone, whereas AG1478 and (−)-blebbistatin (P < 0.0001 versus Ap3A) slowed the migration rate. Western blot assays demonstrated that both dinucleotides activated the ERK1/2 pathway but only Ap4A activated the ROCK-I pathway. The intracellular distribution of pERK1/2 and ROCK-I reflected cross-talk between these two pathways. Conclusions. The activation of the Ap4A/P2Y2 receptor, accelerates corneal epithelial cell migration during wound healing with the activation of MAPK and cytoskeleton pathways, whereas activation of the Ap3A/P2Y6 receptor signals only the MAPK pathway.

The Use of Mucoadhesive Polymers to Enhance the Hypotensive Effect of a Melatonin Analogue, 5-MCA-NAT, in Rabbit Eyes

Purpose.: 5-Methoxy-carbonylamino-N-acetyltryptamine (5-MCA-NAT, a melatonin receptor agonist) produces a clear intraocular pressure (IOP) reduction in New Zealand White rabbits and glaucomatous monkeys. The goal of this study was to evaluate whether the hypotensive effect of 5-MCA-NAT was enhanced by the presence of cellulose derivatives, some of them with bioadhesive properties, as well as to determine whether these formulations were well tolerated by the ocular surface. Methods.: Formulations were prepared with propylene glycol (0.275%), carboxymethyl cellulose (CMC, 0.5% and 1.0%) of low and medium viscosity and hydroxypropylmethyl cellulose (0.3%). Quantification of 5-MCA-NAT (100 μM) was assessed by HPLC. In vitro tolerance was evaluated by the MTT method in human corneal-limbal epithelial cells and normal human conjunctival cells. In vivo tolerance was analyzed by biomicroscopy and specular microscopy in rabbit eyes. The ocular hypotensive effect was evaluated measuring IOP for 8 hours in rabbit eyes. Results.: All the formulations demonstrated good in vitro and in vivo tolerance. 5-MCA-NAT in CMC medium viscosity 0.5% was the most effective at reducing IOP (maximum IOP reduction, 30.27%), and its effect lasted approximately 7 hours. Conclusions.: The hypotensive effect of 5-MCA-NAT was increased by using bioadhesive polymers in formulations that are suitable for the ocular surface and also protective of the eye in long-term therapies. The use of 5-MCA-NAT combined with bioadhesive polymers is a good strategy in the treatment of ocular hypertension and glaucoma.

Diadenosine Polyphosphates in Tears of Sjögren Syndrome Patients

Purpose.: To analyze the levels of diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) in tears of subjects with Sjögren syndrome and to compare them with those in a control group. Methods.: Twelve subjects with a diagnosis of Sjögren syndrome and 20 healthy control subjects were invited to participate in the present study. Schirmer strips were used to measure tear secretion (Schirmer I test) and to collect tears. Ap4A and Ap5A were measured by high-pressure liquid chromatography (HPLC), and a dry eye questionnaire (DEQ) was used to evaluate dry eye symptomatology. Results.: The mean concentrations of Ap4A and Ap5A in the Sjögren syndrome group were 2.54 ± 1.02 and 26.13 ± 6.95 μM, respectively. This group of patients was divided in two subgroups: four patients with normal tear production and eight patients with low tear production. Concentrations of Ap4A, and Ap5A in patients with normal tear production (Schirmer test result, 12.3 ± 1.2 mm) were 0.47 ± 0.20 and 8.03 ± 3.27 μM, respectively. In the patients with low tear production (Schirmer test result, 1.0 ± 0.3 mm), the concentrations were 4.09 ± 1.36 and 39.51 ± 8.46 μM, respectively and in the control group, 0.13 ± 0.03 and 0.04 ± 0.02 μM, respectively. Conclusions.: Patients with Sjögren syndrome have abnormally elevated concentrations of diadenosine polyphosphates, indicating that these compounds could be used in the diagnosis of this disease.

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 P2Y2 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.

Tear Secretion Induced by Selective Stimulation of Corneal and Conjunctival Sensory Nerve Fibers

Purpose. To measure the increase in tear secretion evoked by selective stimulation of the different populations of sensory receptors of the cornea and conjunctiva by using moderate and intense mechanical, chemical, and cold stimuli. Methods. Six healthy subjects participated in the study. Tear secretion was measured in both eyes by the Schirmer’s test conducted under control conditions and after stimulation of the center of the cornea and the temporal conjunctiva with a gas esthesiometer. Mechanical stimulation consisted in three pulses of 3 seconds’ duration of warmed air (at 34°C on the eye surface) applied at moderate (170 mL/min) and high (260 mL/min) flow rates. Cold thermal stimulation was made with cooled air that produced a corneal temperature drop of −1°C or −4.5°C. Chemical (acidic) stimulation was performed with a jet of gas containing a mixture of 80% CO2 in air. Results. The basal volume of tear secretion increased significantly (P < 0.05, paired t-test) after stimulation of the cornea with high-flow mechanical stimuli (260 mL/min), intense cooling pulses (−4.5°C), and chemical stimulation (80% CO2). The same stimuli were ineffective when applied to the conjunctiva. Moderate mechanical (170 mL/min) and cold (−1°C) stimulation of the cornea or the conjunctiva did not change significantly the volume of tear secretion. Conclusions. Reflex tear secretion caused by corneal stimulation seems to be chiefly due to activation of corneal polymodal nociceptors, whereas selective excitation of corneal mechanonociceptors or cold receptors appears to be less effective in evoking an augmented lacrimal secretion. Conjunctival receptors stimulated at equivalent levels do not evoke an increased tear secretion.

Changes in Diadenosine Polyphosphates during Alignment-Fit and Orthokeratology Rigid Gas Permeable

Purpose.: To evaluate the levels of dinucleotides diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) in tears of patients wearing rigid gas permeable (RGP) contact lenses on a daily wear basis and of patients wearing reverse-geometry RGP lenses overnight for orthokeratology treatment. Methods.: Twenty-two young volunteers (10 females, 12 males; 23.47 ± 4.49 years) were fitted with an alignment-fit RGP lens (paflufocon B) for a month, and after a 15-day washout period they were fitted with reverse-geometry RGP lenses for corneal reshaping (paflufocon D) for another month. During each period, tears were collected at baseline day 1, 7, 15, and 28. Ap4A and Ap5A were measured by high-pressure liquid chromatography (HPLC). Additionally, corneal staining, break-up time (BUT), Schirmer test, and dryness symptoms were evaluated. Results.: Ap4A concentrations increased significantly from baseline during the whole period of daily wear of RGP lenses (P < 0.001); concentration was also significantly higher than in the orthokeratology group, which remained at baseline levels during the study period except at day 1 (P < 0.001) and day 28 (P = 0.041). While BUT and Schirmer remained unchanged in both groups, discomfort and dryness were significantly increased during alignment-fit RGP daily wear but not during the orthokeratology period. Conclusions.: Daily wear of RGP lenses increased the levels of Ap4A due to mechanical stimulation by blinking of the corneal epithelium, and this is associated with discomfort. Also, orthokeratology did not produce symptoms or signs of ocular dryness, which could be a potential advantage over soft contact lenses in terms of contact lens-induced dryness.

Contrast sensitivity evaluation with filter contact lenses in patients with retinitis pigmentosa: a pilot study

Purpose: the aim of this pilot study was to test whether retinitis pigmentosa patients would benefit from filter contact lenses as an effective optical aid against glare and photophobia. Methods: fifteen subjects with retinitis pigmentosa were enrolled in this study. All of them were evaluated with filter soft contact lenses (MaxSight), filter glasses (CPF 527) and without filters (control). All patients were assessed for the three aid conditions by means of best corrected visual acuity (BCVA), contrast sensitivity (without glare and with central and peripheral glare)(CSV-1000) and a specific subjective questionnaire about quality of vision. Results: BCVA was slightly better with filters than without filter but the differences were not statistically significant. Contrast sensitivity without glare improved significantly with the contact lenses (p<0.05). The central glare had significant differences for the frequencies of 3 cpd and 18 cpd between the contact lens filter and the control group (p=0.021 and p=0.044, respectively). For the peripheral glare contrast sensitivity improved with contact lens versus control group for highest frequencies, 12 and 18 cpd (p<0.001 and p=0.045, respectively). According to the questionnaire the contact lens filter gave them more visual comfort than the glasses filter under the scenarios of indoors glare, outdoors activities and indoors comfort. Conclusion: the filter contact lenses seem to be a good option to improve the quality of vision of patients with retinitis pigmentosa.

The Impact of the Eye in Dementia: The Eye and its Role in Diagnosis and Follow‐up

Over the last few decades, the importance of ophthalmic examination in neurodegenerative diseases of the CNS has reportedly increased. The retina is an extension of the CNS and thus should not be surprising to find abnormal results in both the test exploring visual processing and those examining the retina of patients with CNS degeneration. Current in vivo imaging techniques are allowing ophthalmologists to detect and quantify data consistent with the histopathological findings described in the retinas of Alzheimer’s disease (AD) patients and may help to reveal unsuspected retinal and optic‐nerve repercussions of other CNS diseases. In this chapter, we perform an analysis of the physiological changes in ocular and cerebral ageing. We analyse the ocular manifestations in CNS disorders such as stroke, AD and Parkinson’s disease. In addition, the pathophysiology of both the eye and the visual pathway in AD are described. The value of the visual psychophysical tests in AD diagnosis is reviewed as well as the main findings of the optical coherence tomography as a contribution to the diagnosis and monitoring of the disease. Finally, we examine the association of two neurodegenerative diseases, AD and glaucoma, as mere coincidence or possible role in the progression of the neurodegeneration.

Macular Thickness as a Potential Biomarker of Mild Alzheimer's Disease

Although several postmortem findings in the retina of patients with Alzheimer's disease (AD) are available, new biomarkers for early diagnosis and follow-up of AD are still lacking. It has been postulated that the defects in the retinal nerve fiber layer (RNFL) may be the earliest sign of AD, even before damage to the hippocampal region that affects memory. This fact may reflect retinal neuronal-ganglion cell death and axonal loss in the optic nerve in addition to aging.

Impaired Mesopic Visual Acuity in Eyes with Early Age-Related Macular Degeneration

Purpose.: To determine photopic and mesopic distance high-contrast visual acuity (HC-VA) and low-contrast visual acuity (LC-VA) in eyes with early age-related macular degeneration (AMD). Methods.: Measurements were made in 22 subjects with early AMD and 28 healthy control subjects. Inclusion criteria included a photopic HC-VA of 20/25 or better. Distance VA was measured using HC (96%) and LC (10%) Bailey-Lovie logMAR letter charts under photopic (85 cd/m2) and mesopic (0.1–0.2 cd/m2) luminance conditions. Results.: Mean mesopic distance HC-VA and LC-VA were significantly worse (0.1 logMAR and 0.28 logMAR, respectively) in the early AMD group than in the control group. Under mesopic conditions, the mean difference between LC-VA and HC-VA was significantly greater in the early AMD (0.45 logMAR) than the control group (0.27 logMAR). Mean differences between mesopic versus photopic HC-VA and mesopic versus photopic LC-VA were significantly greater in the early AMD than the control group (0.13 and 0.32 logMAR of difference between the means, respectively). Sensitivity and specificity were significantly greater for mesopic LC-VA than for mesopic HC-VA (Receiver Operating Characteristics, area under the curve [AUC], 0.94 ± 0.030 and 0.76 ± 0.067, respectively). AUC values for photopic HC-VA and LC-VA were below 0.70. Conclusions.: Visual acuity testing under low luminance conditions emerged as an optimal quantitative measure of retinal function in early AMD.