Caracterización de un modelo murino de glaucoma: implicación funcional de la señalización del receptor P2X7 en procesos neurodegenerativos de la retina

El glaucoma es una de las causas más comunes de discapacidad visual y una de las enfermedades neurodegenerativas oculares más frecuentes de pérdida irreversible de visión. La afectación originada en la retina se caracteriza por la degeneración de las células ganglionares y la pérdida de axones. La presión intraocular es un factor de riesgo importante en el glaucoma, entre otros factores, implicando mecanismos bioquímicos que desencadenan la muerte de las células ganglionares. El ratón DBA/2J es un modelo de hipertensión ocular y de degeneración de las células ganglionares de la retina (CGR). Las características principales de éste son la dispersión del pigmento del iris (IPD) y la atrofia del estroma del iris (ISA) que conducen a la patogénesis del glaucoma. Los mecanismos bioquímicos que comprometen al sistema purinérgico en procesos patológicos como la degeneración glaucomatosa han sido estudiados en los últimos años, siendo de gran relevancia como posibles dianas farmacológicas para el tratamiento de diferentes neuropatías. Los receptores P2X comprenden una familia de siete canales iónicos de membrana activados por ligando (P2X1-7) que se activan por el ATP extracelular (ATPe). En particular, los receptores P2X7 podrían estar involucrados en la regulación de la transmisión sináptica y la muerte neuronal en la retina. Además, la excitotoxicidad mediada por ATP a través de la activación del receptor P2X7 sugiere su posible implicación en la degeneración neuronal y la pérdida de la función visual en las retinas glaucomatosas. Tan importante como la presencia de este receptor purinérgico es estudiar los niveles de ATP extracelular de la retina, así como evaluar los cambios en la expresión del transportador de nucleótidos vesicular (VNUT) y los niveles de ecto-nucleotidasa (E-NPP1) en este modelo murino de glaucoma durante el desarrollo de la enfermedad...

Estudio a corto y largo plazo de la melatonina y el 5-MCA-NAT como tratamiento en la hipertensión ocular y el glaucoma

El glaucoma engloba una serie de enfermedades que se caracterizan por la pérdida progresiva de las células ganglionares de la retina y la neuropatía del nervio óptico, que desencadenan finalmente una ceguera irreversible. La elevación de la presión intraocular (PIO) aparece como el principal desencadenante de esta patología en la mayoría de los casos, ya que produce una disminución del flujo sanguíneo hacia la retina y un aumento del estrés oxidativo. La estrategia terapéutica más común implica el uso de fármacos hipotensores, a menudo combinados, asociados a efectos no deseados. En este marco, la melatonina y sus análogos, como el 5-MCA-NAT, surgen como alternativas a los fármacos ya existentes, por sus propiedades hipotensoras y antioxidantes y, en el caso del 5-MCA-NAT, por su posible acción a largo plazo. El objetivo de esta tesis es elucidar los mecanismos de la acción hipotensora aguda ya descrita para estos compuestos, determinando además el receptor y las vías de señalización implicadas en esta respuesta a corto plazo. En este mismo sentido nos proponemos confirmar la existencia de efectos hipotensores a largo plazo, analizando esta respuesta a diferentes fármacos y esclareciendo si ésta se debe a la capacidad de estos compuestos como modificadores de la expresión génica de enzimas o receptores implicados en la regulación de la PIO...

Mecanismos celulares activados por receptores P2Y₂ en los procesos ciliares: implicaciones en la hipertensión ocular y el glaucoma

El glaucoma es una neuropatía óptica crónica de etiología multifactorial, que provoca una pérdida progresiva e irreversible de la visión debida a la degeneración de los axones del nervio óptico y la muerte apoptótica de las células ganglionares de la retina (Downs et al., 2011; Quigley, 2011; Soto et al., 2014). Actualmente es la principal causa de ceguera irreversible en el mundo (Pascolini et al., 2012). Aunque su etiología aún no está clara, se sabe que el principal factor de riesgo para desarrollar la enfermedad es el aumento de la PIO debido a anomalías en la dinámica del humor acuoso (Casson et al., 2012). La presencia de receptores purinérgicos P2X y P2Y ha sido descrita en las principales estructuras oculares relacionadas con la producción y drenaje del humor acuoso, los procesos ciliares y la malla trabecular, que son bañadas por dicho fluido (Guzman-­‐Aranguez et al., 2013). Al mismo tiempo, se ha referido la presencia del dinucleótido Ap4A, agonista de dichos receptores, en el humor acuoso, y cómo sus niveles se veían significativamente incrementados en pacientes glaucomatosos (Castany et al., 2011). El objetivo de esta tesis ha sido evaluar la implicación de los receptores purinérgicos P2Y2 de los procesos ciliares en la hipertensión ocular. Más concretamente determinar el papel del Ap4A a través de la activación de dichos receptores sobre la movilización del ion cloruro y del agua a través de las proteínas acuaporinas (AQPs), principales componentes del humor acuoso. Por otra parte, se ha investigado el origen de los niveles incrementados de Ap4A debidos al aumento de la PIO, caracterizando uno de los posibles sensores celulares implicados en dicho proceso. Al mismo tiempo se han evaluado y caracterizado los diferentes mecanismos implicados en dicha liberación...

A Diagnostic Calculator for Detecting Glaucoma on the Basis of Retinal Nerve Fiber Layer, Optic Disc, and Retinal Ganglion Cell Analysis by Optical Coherence Tomography

Purpose: The purpose of this study was to develop and validate a multivariate predictive model to detect glaucoma by using a combination of retinal nerve fiber layer (RNFL), retinal ganglion cell-inner plexiform (GCIPL), and optic disc parameters measured using spectral-domain optical coherence tomography (OCT). Methods: Five hundred eyes from 500 participants and 187 eyes of another 187 participants were included in the study and validation groups, respectively. Patients with glaucoma were classified in five groups based on visual field damage. Sensitivity and specificity of all glaucoma OCT parameters were analyzed. Receiver operating characteristic curves (ROC) and areas under the ROC (AUC) were compared. Three predictive multivariate models (quantitative, qualitative, and combined) that used a combination of the best OCT parameters were constructed. A diagnostic calculator was created using the combined multivariate model. Results: The best AUC parameters were: inferior RNFL, average RNFL, vertical cup/disc ratio, minimal GCIPL, and inferior-temporal GCIPL. Comparisons among the parameters did not show that the GCIPL parameters were better than those of the RNFL in early and advanced glaucoma. The highest AUC was in the combined predictive model (0.937; 95% confidence interval, 0.911–0.957) and was significantly (P = 0.0001) higher than the other isolated parameters considered in early and advanced glaucoma. The validation group displayed similar results to those of the study group. Conclusions: Best GCIPL, RNFL, and optic disc parameters showed a similar ability to detect glaucoma. The combined predictive formula improved the glaucoma detection compared to the best isolated parameters evaluated. The diagnostic calculator obtained good classification from participants in both the study and validation groups.

Macro- and microglial responses in the fellow eyes contralateral to glaucomatous eyes

Most studies employing experimental models of unilateral glaucoma have used the normotensive contralateral eye as the normal control. However, some studies have recently reported the activation of the retinal macroglia and microglia in the uninjured eye, suggesting that the eye contralateral to experimental glaucoma should not be used as a control. This review analyzes the studies describing the contralateral findings and discusses some of the routes through which the signals can reach the contralateral eye to initiate the glial reactivation.

Influencia del tratamiento farmacológico hipotensor ocular en la flora bacteriana conjuntival

La flora bacteriana conjuntival desempeña un papel muy relevante en la patogenia de la endoftalmitis postoperatoria. Esta complicación, aunque es infrecuente, tiene una gran importancia dadas sus graves consecuencias sobre la función visual. El conocimiento de las características de la población bacteriana puede permitir establecer tanto valoraciones de riesgo individualizado como terapias profilácticas adaptadas al perfil específico de cada caso. En la situación particular del ojo con glaucoma este hecho adquiere una significación especial por varios factores, a menudo coexistentes: la alteración del complejo película lagrimal-superficie ocular, la habitual necesidad de instilación tópica de fármacos hipotensores de manera crónica, la posible existencia de una conjuntiva alterada por procedimientos quirúrgicos previos incluida la existencia de una ampolla de filtración y, finalmente, la grave repercusión de una contaminación bacteriana sobre un ojo operado de glaucoma. La alteración crónica de la superficie ocular es frecuente en pacientes con glaucoma. La instilación repetida y mantenida de colirios hipotensores, cuyos principios activos y conservantes pueden alterar la película lagrimal y dar lugar a modificaciones histológicas de la conjuntiva, puede reducir la capacidad de defensa del ojo frente a la agresión bacteriana...

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.

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.

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.

Retinal Macroglial Responses in Health and Disease

Due to their permanent and close proximity to neurons, glial cells perform essential tasks for the normal physiology of the retina. Astrocytes andM¨uller cells (retinal macroglia) provide physical support to neurons and supplement them with several metabolites and growth factors.Macroglia are involved in maintaining the homeostasis of extracellular ions and neurotransmitters, are essential for information processing in neural circuits, participate in retinal glucose metabolism and in removing metabolic waste products, regulate local blood flow, induce the blood-retinal barrier (BRB), play fundamental roles in local immune response, and protect neurons from oxidative damage. In response to polyetiological insults, glia cells react with a process called reactive gliosis, seeking to maintain retinal homeostasis. When malfunctioning, macroglial cells can become primary pathogenic elements. A reactive gliosis has been described in different retinal pathologies, including age-related macular degeneration (AMD), diabetes, glaucoma, retinal detachment, or retinitis pigmentosa. A better understanding of the dual, neuroprotective, or cytotoxic effect of macroglial involvement in retinal pathologies would help in treating the physiopathology of these diseases.The extensive participation of the macroglia in retinal diseases points to these cells as innovative targets for new drug therapies.

Melatonin Makes Me Feel Awake! Seven Years of Lab Experience (2000-2007)

The research on the effect of melatonin on intraocular pressure (IOP) is reviewed from the hystorical point of view of our laboratory. The original idea of melatonin modulating intraocular pressure has been improved by using selective compounds for MT2 and specially melatonin MT3 receptors. The selective compound 5-methoxyamino N-acetyltryptamine (5-MCA-NAT) has been an attractive compound due to its ability to reduce IOP about 40%, therefore being a good candidate to the treatment of the ocular hypertension linked to glaucoma. More compounds have been developed and tested permitting us to have a more accurate panorama of those receptors controlling the relevant process of intraocular pressure.