Papel biológico del diadenosin tetrafosfato en el ojo: efecto sobre la composición lacrimal e implicación en la función de barrera corneal

El Ap4A es una molécula con un amplio papel biológico en el ojo. Se ha descrito su implicación en procesos de secreción lagrimal, cicatrización epitelial, regulación de la presión intraocular, y presenta también un papel neuroprotector sobre los terminales simpáticos que inervan el cuerpo ciliar. El objetivo de este trabajo ha sido analizar la participación del Ap4A en otras posibles funciones a nivel ocular. En concreto se ha estudiado la capacidad de este dinucléotido para estimular la liberación de proteínas lagrimales de acción antibacteriana tales como la lisozima y la lactoferrina. Por otra parte se ha investigado su efecto como modulador de la función de barrera de la córnea a través de la regulación de los niveles de expresión de proteínas constituyentes de las tight junctions (TJ). Dicho efecto sobre la barrera puede tener una importante repercusión en la entrada de fármacos y en la consiguiente eficacia terapéutica de los mismos. Por último, se ha estudiado la posible implicación del dinucleótido en el proceso de edematización descrito en modelos animales glaucomatosos tales como el ratón DBA/2J. Con el objetivo de averiguar si la activación de Ap4A inducía un efecto sobre la producción de dos proteínas antimicrobianas relevantes de la lágrima (lisozima y lactoferrina) se realizaron ensayos en la lágrima de conejos albinos de Nueva Zelanda, mediante las técnicas de agar-agar y ELISA. Los resultados obtenidos demostraron que Ap4A produce un aumento en la concentración de lisozima y de lactoferrina del 93% y 24%, respectivamente, frente a valores basales, y este efecto está mediado por receptores de tipo P2...

Caracterización de la citología de impresión corneal en la especie canina y su aplicación en el diagnóstico de la queratoconjuntivitis seca (QCS)

La citología de impresión corneal es una técnica atraumática, simple, rápida y carente de efectos secundarios que permite estudiar las capas celulares más externas de la superficie ocular, posibilitando el diagnóstico de enfermedades de la misma y su monitorización durante y después del tratamiento. Los objetivos de la presente Tesis Doctoral han sido normalizar la técnica de obtención y procesamiento de muestras obtenidas por citología de impresión corneal en la especie canina, determinar los parámetros morfológicos y morfométricos fisiológicos de las células epiteliales corneales en perros sanos y establecer las alteraciones celulares corneales en el transcurso de la QCS canina. Además, se ha estudiado la existencia de una correlación entre las alteraciones del epitelio corneal y la producción lagrimal valorada mediante la prueba lagrimal de Schirmer I (PLS I). En este estudio se han incluido 60 perros de diferentes razas que han sido divididos en dos poblaciones: el grupo I, formado por pacientes sanos, con unos valores en la prueba lagrimal de Schirmer I (PLS I) iguales o superiores a 15 mm/min y el grupo II, formado por pacientes diagnosticados de QCS con unos valores en la PLS I comprendidos entre 0 y 14 mm/min. Los ojos de los animales del grupo II han sido categorizados a su vez en 3 subgrupos, estableciendo la gravedad de la enfermedad en función de los resultados obtenidos en la PLS I como grave, moderada o leve. Para facilitar la recogida y el manejo de las muestras, adecuar la técnica para su empleo en animales y disminuir al mínimo cualquier tipo de lesión iatrogénica sobre la superficie ocular se ha utilizado un dispositivo simple y eficaz, diferente a los que se han venido empleando tradicionalmente en oftalmología humana y veterinaria. La valoración morfométrica de las células se ha realizado atendiendo a los criterios de celularidad de la muestra, separación intercelular, morfología de las células, tinción citoplasmática, área celular, área nuclear, ratio núcleo/citoplasma (ratio N:C) y alteraciones nucleares...

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.

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.

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.

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.

Cell Pattern in Adult Human Corneal Endothelium

A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.