Neuroprotective effects of the cannabinoid agonist HU210 on retinal degeneration

Cannabinoids have been demonstrated to exert neuroprotective effects on different types of neuronal insults. Here we have addressed the therapeutic potential of the synthetic cannabinoid HU210 on photoreceptor degeneration, synaptic connectivity and functional activity of the retina in the transgenic P23H rat, an animal model for autosomal dominant retinitis pigmentosa (RP). In P23H rats administered with HU210 (100 μg/kg, i.p.) from P24 to P90, ERG recordings showed an amelioration of vision loss, as compared to vehicle-administered animals. Under scotopic conditions, the maximum a-wave amplitudes recorded at P60 and P90 were higher in HU210-treated animals, as compared to the values obtained in untreated animals. The scotopic b-waves were significantly higher in treated animals than in untreated rats at P30, P60 and P90. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. HU210-treated animals had 40% more photoreceptors than untreated animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were also preserved in HU210-treated P23H rats. These results indicate that HU210 preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in P23H rats. These data suggest that cannabinoids are potentially useful to delay retinal degeneration in RP patients.

In vitro differentiation of murine hematopoietic progenitor cells toward the myeloid lineage occurs in response to Staphylococcus aureus and yeast species

We have studied the effect of inactivated microbial stimuli (Candida albicans, Candida glabrata, Saccharomyces boulardii, and Staphylococcus aureus) on the in vitro differentiation of lineage negative (Lin−) hematopoietic progenitor mouse cells. Purified Lin− progenitors were co-cultured for 7 days with the stimuli, and cell differentiation was determined by flow cytometry analysis. All the stimuli assayed caused differentiation toward the myeloid lineage. S. boulardii and particularly C. glabrata were the stimuli that induced in a minor extent differentiation of Lin− cells, as the major population of differentiated cells corresponded to monocytes, whereas C. albicans and S. aureus induced differentiation beyond monocytes: to monocyte-derived dendritic cells and macrophages, respectively. Interestingly, signaling through TLR2 by its pure ligand Pam3CSK4 directed differentiation of Lin− cells almost exclusively to macrophages. These data support the notion that hematopoiesis can be modulated in response to microbial stimuli in a pathogen-dependent manner, being determined by the pathogen-associated molecular patterns and the pattern-recognition receptors involved, in order to generate the populations of mature cells required to deal with the pathogen.

Choroidal Thickness And Volume In Healthy Young Whites And Their Relationship With Axial Length, Ammetropy And Sex

Purpose: To evaluate choroidal thickness in young subjects using Enhanced Depth Imaging Spectral Domain Optical Coherence Tomography (EDI SD-OCT) describing volume differences between all the defined areas of the Early Treatment Diabetic Retinopathy Study (ETDRS). Design: Prospective, clinical study. Methods: Seventy-nine eyes of 95 healthy, young (23.8±3.2years), adult volunteers were prospectively enrolled. Manual choroidal segmentation on a 25-raster horizontal scan protocol was performed. The measurements of the nine subfields defined by the ETDRS were evaluated. Results: Mean subfoveal choroidal thickness was 345.67±81.80μm and mean total choroidal volume was 8.99±1.88mm3. Choroidal thickness and volume were higher at the superior and temporal areas compared to inferior and nasal sectors of the same diameter respectively. Strong correlations between subfoveal choroidal thickness and axial length (AL) and myopic refractive error were obtained, r = -0.649, p<0.001 and r = 0.473, p<0.001 respectively. Emmetropic eyes tended to have thicker subfoveal choroidal thickness (381.94±79.88μm versus 307.04±64.91μm) and higher total choroidal volume than myopic eyes (9.80± 1.87mm3 versus 8.14±1.48mm3). The estimation of the variation of the subfoveal choroidal thickness with the AL was-43.84μm/mm. In the myopic group, the variation of the subfoveal choroidal thickness with the myopic refractive error was -10.45μm/D. Conclusions: This study establishes for the first time a normal database for choroidal thickness and volume in young adults. Axial length, and myopic ammetropy are highly associated with choroidal parameters in healthy subjects. EDI SD-OCT exhibited a high degree of intraobserver and interobserver repeatability.

Characterization of a new murine retinal cell line (MU-PH1) with glial, progenitor and photoreceptor characteristics

Unlike fish and amphibians, mammals do not regenerate retinal neurons throughout life. However, neurogenic potential may be conserved in adult mammal retina and it is necessary to identify the factors that regulate retinal progenitor cells (RPC) proliferative capacity to scope their therapeutic potential. Müller cells can be progenitors for retinal neuronal cells and can play an essential role in the restoration of visual function after retinal injury. Some members of the Toll-like receptor (TLR) family, TLR2, TLR3 and TLR4, are related to progenitor cells proliferation. Müller cells are important in retinal regeneration and stable cell lines are useful for the study of retinal stem cell biology. Our purpose was to obtain a Müller-derived cell line with progenitor characteristics and potential interest in regeneration processes. We obtained and characterized a murine Müller-derived cell line (MU-PH1), which proliferates indefinitely in vitro. Our results show that (i) MU-PH1 cells expresses the Müller cell markers Vimentin, S-100, glutamine synthetase and the progenitor and stem cell markers Nestin, Abcg2, Ascl1, α-tubulin and β-III-tubulin, whereas lacks the expression of CRALBP, GFAP, Chx10, Pax6 and Notch1 markers; (ii) MU-PH1 cell line stably express the photoreceptor markers recoverin, transducin, rhodopsin, blue and red/green opsins and also melanopsin; (iii) the presence of opsins was confirmed by the recording of intracellular free calcium levels during light stimulation; (iv) MU-PH1 cell line also expresses the melatonin MT1 and MT2 receptors; (v) MU-PH1 cells express TLR1, 2, 4 and 6 mRNA; (vi) MU-PH1 express TLR2 at cell surface level; (vii) Candida albicans increases TLR2 and TLR6 mRNA expression; (viii) C. albicans or TLR selective agonists (Pam(3)CysSK(4), LPS) did not elicit morphological changes nor TNF-α secretion; (ix) C. albicans and Pam(3)CysSK(4) augmented MU-PH1 neurospheres formation in a statistically significant manner. Our results indicate that MU-PH1 cell line could be of great interest both as a photoreceptor model and in retinal regeneration approaches and that TLR2 may also play a role in retinal cell proliferation.

Impairment of Intrinsically Photosensitive Retinal Ganglion Cells Associated With Late Stages of Retinal Degeneration

Purpose. To evaluate quantitative and qualitative age-related changes in intrinsically photosensitive melanopsin-containing retinal ganglion cells (ipRGCs) in transgenic P23H rats, an animal model of autosomal dominant retinitis pigmentosa (RP) was examined. Methods. ipRGC density, morphology, and integrity were characterized by immunohistochemistry in retinas extracted from P23H and Sprague–Dawley (SD) rats aged 4, 12, and 18 months. Differences between SD and P23H rats throughout the experimental stages, as well as the interactions among them, were morphologically evaluated. Results. In rat retinas, we have identified ipRGCs with dendrites stratifying in either the outer margin (M1) or inner side (M2) of the inner plexiform layer, and in both the outer and inner plexuses (M3). A small group of M1 cells had their somas located in the inner nuclear layer (M1d). In SD rats, ipRGCs showed no significant changes associated with age, in terms of either mean cell density or the morphologic parameters analyzed. However, the mean density of ipRGCs in P23H rats fell by approximately 67% between 4 and 18 months of age. Moreover, ipRGCs in these animals showed a progressive age-dependent decrease in the dendritic area, the number of branch points and terminal neurite tips per cell, and the Sholl area. Conclusions. In the P23H rat model of retinitis pigmentosa, density, wholeness, and dendritic arborization of melanopsin-containing ganglion cells decrease in advanced stages of the degenerative disease.

Changes in the Photoreceptor Mosaic of P23H-1 Rats During Retinal Degeneration: Implications for Rod-Cone Dependent Survival

Purpose. To investigate the spatiotemporal relationship between rod and cone degeneration in the P23H-1 rat. Methods. Control Sprague-Dawley (SD) and P23H-1 rats of ages ranging from P30 to P365 were used. Retinas were processed for whole mounts or cross sections and rods and cones were immunodetected. We used newly developed image analysis techniques to quantify the total population of L/M cones (the most abundant cones in the rat) and analyzed the rings of rod-cone degeneration. Results. In P23H-1 rats, rod degeneration occurs rapidly: first the rod outer segment shortens, at P30 there is extensive rod loss, and by P180 rod loss is almost complete except for the most peripheral retina. The numbers of L/M cones are, at all postnatal ages, lower in P23H-1 rats than in control SD rats, and decrease significantly with age (by P180). Rod and cone degeneration is spatiotemporally related and occurs in rings that appear already at P90 and spread throughout the entire retina. At P180, the rings of rod-cone degeneration are more abundant in the equatorial retina and are larger in the dorsal retina. Conclusions. This work describes for the first time that in the P23H-1 rat, rod and cone degeneration is spatiotemporally related and occurs in rings. Cone loss follows rod loss and starts very soon, even before P30, the first age analyzed here. The characteristics of the rings suggest that secondary cone degeneration is influenced by retinal position and/or other intrinsic or extrinsic factors.

Phagocytosis of Photoreceptor Outer Segments by Transplanted Human Neural Stem Cells as a Neuroprotective Mechanism in Retinal Degeneration

Purpose. Transplantation of human central nervous system stem cells (HuCNS-SC) into the subretinal space of Royal College of Surgeons (RCS) rats preserves photoreceptors and visual function. To explore possible mechanism(s) of action underlying this neuroprotective effect, we performed a detailed morphologic and ultrastructure analysis of HuCNS-SC transplanted retinas. Methods. The HuCNS-SC were transplanted into the subretinal space of RCS rats. Histologic examination of the transplanted retinas was performed by light and electron microscopy. Areas of the retina adjacent to HuCNS-SC graft (treated regions) were analyzed and compared to control sections obtained from the same retina, but distant from the transplant site (untreated regions). Results. The HuCNS-SC were detected as a layer of STEM 121 immunopositive cells in the subretinal space. In treated regions, preserved photoreceptor nuclei, as well as inner and outer segments were identified readily. In contrast, classic signs of degeneration were observed in the untreated regions. Interestingly, detailed ultrastructure analysis revealed a striking preservation of the photoreceptor–bipolar–horizontal cell synaptic contacts in the outer plexiform layer (OPL) of treated areas, in stark contrast with untreated areas. Finally, the presence of phagosomes and vesicles exhibiting the lamellar structure of outer segments also was detected within the cytosol of HuCNS-SC, indicating that these cells have phagocytic capacity in vivo. Conclusions. This study reveals the novel finding that preservation of specialized synaptic contacts between photoreceptors and second order neurons, as well as phagocytosis of photoreceptor outer segments, are potential mechanism(s) of HuCNS-SC transplantation, mediating functional rescue in retinal degeneration.

Electroretinographical and histological study of mouse retina after optic nerve section: a comparison between wild-type and retinal degeneration 1 mice

Background: Retinal ganglion cell death underlies the pathophysiology of neurodegenerative disorders such as glaucoma or optic nerve trauma. To assess the potential influence of photoreceptor degeneration on retinal ganglion cell survival, and to evaluate functionality, we took advantage of the optic nerve section mouse model. Methods: Surviving retinal ganglion cells were double-stained by exposing both superior colliculi to fluorogold, and by applying dextran-tetramethylrhodamine to the injured optic nerve stump. To assess retinal function in wild-type animals, electroretinograms were recorded on the injured eyes and compared with the contralateral. Similar labelling experiments were carried out on retinal degeneration 1 mice. Surviving retinal ganglion cells were counted 21 days after axotomy and compared with wild-type mice. No functional experiments were performed on retinal degeneration 1 animals because they do not develop normal electroretinographical responses. Results: A significant decrease in retinal ganglion cell density was observed 6 days after axotomy in the wild type. Functional studies revealed that, in scotopic conditions, axotomy induced a significant amplitude decrease in the positive scotopic threshold response component of the electroretinogram. Such decrease paralleled cell loss, suggesting it may be an appropriate technique to evaluate functionality. When comparing retinal ganglion cell densities in wild-type and retinal degeneration 1 mice, a significant greater survival was observed on the latter. Conclusions: After optic nerve section, electroretinographical recordings exhibited a progressive decrease in the amplitude of the positive scotopic threshold response wave, reflecting ganglion cell loss. Interestingly, rod degeneration seemed, at least initially, to protect from axotomy-driven damage.

Candida albicans stimulates in vivo differentiation of haematopoietic stem and progenitor cells towards macrophages by a TLR2-dependent signalling

Toll-like receptors (TLRs) are expressed by haematopoietic stem and progenitor cells (HSPCs), and may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that (i) inactivated yeasts of Candida albicans induce in vitro differentiation of HSPCs towards the myeloid lineage, and (ii) soluble TLR agonists induce in vivo their differentiation towards macrophages. In this work, using an in vivo model of HSPCs transplantation, we report for the first time that HSPCs sense C. albicans in vivo and subsequently are directed to produce macrophages by a TLR2-dependent signalling. Purified lineage-negative cells (Lin−) from bone marrow of C57BL/6 mice (CD45.2 alloantigen) were transplanted into B6Ly5.1 mice (CD45.1 alloantigen), which were then injected with viable or inactivated C. albicans yeasts. Transplanted cells were detected in the spleen and in the bone marrow of recipient mice, and they differentiate preferentially to macrophages, both in response to infection or in response to inactivated yeasts. The generation of macrophages was dependent on TLR2 but independent of TLR4, as transplanted Lin− cells from TLR2−/− mice did not give rise to macrophages, whereas Lin− cells from TLR4−/− mice generated macrophages similarly to control cells. Interestingly, the absence of TLR2, or in a minor extent TLR4, gives Lin− cells an advantage in transplantation assays, as increases the percentage of transplanted recovered cells. Our results indicatethat TLR-mediated recognition of C. albicans by HSPCs may help replace and/or increase cells that constitute the first line of defence against the fungus, and suggest that TLR-mediated signalling may lead to reprogramming early progenitors to rapidly replenishing the innate immune system and generate the most necessary mature cells to deal with the pathogen.

Physiological and Morphological Similarities between the Octodon Degus Retina and the Human Retina

Octodon degus is a rodent with diurnal crepuscular activity. Here we compare the function and morphology of the retina of this diurnal rodent with the retinas of nocturnal rodents and humans.

Loss of Outer Retinal Neurons and Circuitry Alterations in the DBA/2J Mouse

Purpose. The DBA/2J mouse line develops essential iris atrophy, pigment dispersion, and glaucomatous age-related changes, including an increase of IOP, optic nerve atrophy, and retinal ganglion cell (RGC) death. The aim of this study was to evaluate possible morphological changes in the outer retina of the DBA/2J mouse concomitant with disease progression and aging, based on the reduction of both the a- and b-waves and photopic flicker ERGs in this mouse line. Methods. Vertically sectioned DBA/2J mice retinas were evaluated at 3, 8, and 16 months of age using photoreceptor, horizontal, and bipolar cell markers. Sixteen-month-old C57BL/6 mice retinas were used as controls. Results. The DBA/2J mice had outer retinal degeneration at all ages, with the most severe degeneration in the oldest retinas. At 3 months of age, the number of photoreceptor cells and the thickness of the OPL were reduced. In addition, there was a loss of horizontal and ON-bipolar cell processes. At 8 months of age, RGC degeneration occurred in patches, and in the outer retina overlying these patches, cone morphology was impaired with a reduction in size as well as loss of outer segments and growth of horizontal and bipolar cell processes into the outer nuclear layer. At 16 months of age, connectivity between photoreceptors and horizontal and bipolar cell processes overlying these patches was lost. Conclusions. Retinal degeneration in DBA/2J mice includes photoreceptor death, loss of bipolar and horizontal cell processes, and loss of synaptic contacts in an aging-dependent manner.

Uso de material audiovisual como apoyo en las clases teóricas

La disponibilidad de nuevas herramientas multimedia favorece la generación de material docente auxiliar para la enseñanza de las diversas materias y puede suponer un aliciente añadido para el estudiante. Así, además de fomentar su interés por la materia, se puede potenciar su aprendizaje autónomo. Con el objetivo de evaluar las ventajas de la visualización de vídeos como apoyo a las explicaciones teóricas se planteó la siguiente experiencia: con alumnos de la asignatura "Farmacología", del Grado en Óptica y Optometría, se analizó y comparó la comprensión de la materia con y sin la visualización de vídeos como material de apoyo. El análisis mediante cuestionarios mostró que la visualización ayudó a fijar los conocimientos previamente adquiridos. El 79% de los estudiantes consideró que los vídeos les ayudaron a conseguir una mejor comprensión de la materia. El 95% de los alumnos consideró más útil escuchar primero la explicación del docente. El profesorado implicado manifiesta la dificultad de la selección de material óptimo debido su escasez, por lo que resulta conveniente la elaboración de material específico. Podemos concluir que el material audiovisual supone una buena herramienta complementaria para las clases teóricas, si bien debe integrarse de forma adecuada en el desarrollo de la clase.

Trabajo de coordinación para la implantación del Máster en Optometría Avanzada y Salud Visual

Durante el curso 2013-2014 la Universidad de Alicante ha propuesto la implantación del Master en Optometría Avanzada y Salud Visual, dicha solicitud está siendo actualmente evaluada por la ANECA. Con el fin de coordinar la docencia de este Máster y dentro del Proyecto de Redes de Investigación en Docencia Universitaria 2013-2014, se ha creado una red formada por todos los profesores que han participado en la elaboración del plan de estudios. En esta red esta red se pretende la coordinación entre las distintas asignaturas para elaborar las guías docentes a partir de los datos de las fichas enviadas a la ANECA. Por otra parte también se ha modificado la memoria atendiendo a las alegaciones realizadas por la ANECA. Y se han desarrollado los contenidos, la metodología de las distintas actividades propuestas con el fin de asegurar la consecución de las competencias previstas.

Experiencia de trabajo colaborativo internacional para la elaboración de material docente

El aprendizaje basado en problemas (ABP) se viene utilizando desde hace años como estrategia de enseñanza porque fomenta el autoaprendizaje y está bien valorado por los estudiantes. El objetivo de este trabajo fue crear una colección de problemas que sea de utilidad como recurso didáctico para el estudio de la Farmacología. Para ello se preparó una colección de problemas sobre alteraciones del comportamiento inducidas por fármacos, para ser utilizados en tres universidades iberoamericanas: Universidad de Alicante (España), Universidad Nacional de Tucumán (Argentina) y Universidad de Montevideo (Uruguay). Los problemas fueron elaborados de forma colaborativa por profesores de las tres universidades; fueron analizados por expertos en diferentes materias (psiquiatría, derecho, sociología) y después fueron aplicados a nivel grado (Criminología, Universidad de Alicante y Odontología, Universidad de Tucumán) y postgrado (Universidad de Montevideo). La calidad del aprendizaje se midió de forma cuantitativa (grado de acierto a las preguntas que se realizaban después de cada problema) y de forma cualitativa (valoración de las actitudes mediante encuesta con escala Likert). En todos los casos los problemas demostraron ser de utilidad en las tres universidades y fueron bien valorados por el profesorado, los expertos y los estudiantes.

Microglia activation in a model of retinal degeneration and TUDCA neuroprotective effects

Background: Retinitis pigmentosa is a heterogeneous group of inherited neurodegenerative retinal disorders characterized by a progressive peripheral vision loss and night vision difficulties, subsequently leading to central vision impairment. Chronic microglia activation is associated with various neurodegenerative diseases including retinitis pigmentosa. The objective of this study was to quantify microglia activation in the retina of P23H rats, an animal model of retinitis pigmentosa, and to evaluate the therapeutic effects of TUDCA (tauroursodeoxycholic acid), which has been described as a neuroprotective compound. Methods: For this study, homozygous P23H line 3 and Sprague-Dawley (SD) rats were injected weekly with TUDCA (500 mg/kg, ip) or vehicle (saline) from 20 days to 4 months old. Vertical retinal sections and whole-mount retinas were immunostained for specific markers of microglial cells (anti-CD11b, anti-Iba1 and anti-MHC-II). Microglial cell morphology was analyzed and the number of retinal microglial was quantified. Results: Microglial cells in the SD rat retinas were arranged in regular mosaics homogenously distributed within the plexiform and ganglion cell layers. In the P23H rat retina, microglial cells increased in number in all layers compared with control SD rat retinas, preserving the regular mosaic distribution. In addition, a large number of amoeboid CD11b-positive cells were observed in the P23H rat retina, even in the subretinal space. Retinas of TUDCA-treated P23H animals exhibited lower microglial cell number in all layers and absence of microglial cells in the subretinal space. Conclusions: These results report novel TUDCA anti-inflammatory actions, with potential therapeutic implications for neurodegenerative diseases, including retinitis pigmentosa.