Glutamate transporter localization does not correspond to the temporary functional recovery and late degeneration after acute ocular ischemia in rats

Purpose: To determine whether the localization of retinal glutamate transporters is affected by retinal ischaemia and whether their ability to transport glutamate decreases with the progression of ischemic retinal and optic nerve degeneration. Methods: Retinal ischemia was induced in rats by acutely increasing the intraocular pressure (IOP, 110 mmHg/60 min). Reperfusion was permitted for periods up to 60 days post-ischemia. Functional evaluation was performed by monitoring the pupil light reflexes (PLRs) and electroretinograms (flash, flicker ERG and oscillatory potentials). Glutamate transporter localization and D-aspartate (glutamate analogue) uptake were assessed by immunohistochemistry. Results: Intense immunoreactivity for the retinal glutamate transporters (GLAST, GLT1, EAAC1 and EAAT5) was observed at all time points after the insult, despite severe retinal degeneration. D-aspartate was also normally accumulated in the ischemic retinas. Ten days post-operatively the PLR ratio (ratio = indirect/direct PLR = 34 +/- 7(.)5%) was significantly less than the pre-operative value (pre-op = 76(.)7 +/- 2 (.)6%, p < 0(.)05). However, 25 and 35 days post-operatively PLR ratios did not differ significantly from pre-operative values (44(.)4 +/- 6(.)9 and 53(.)8 +/- 9(.)6%, p > 0(.)05). Forty-five and 60 days post-operatively the PLR ratio declined again and was significantly lower than the pre-operative value (33(.)8 + 8(.)7 and 26(.)2 + 8(.)9%, p < 0(.)05). Statistical analysis revealed that all tested ERG components had significantly higher values at 32, but not at 42 and 58 days post-operatively when compared to the first time point recorded post-operatively (10 days). Conclusions: While retinal glutamate transport is compromised during an acute ischemic insult, consequent retinal recovery and degeneration are not due to a change in the excitatory amino acid transporter localization or D-aspartate (glutamate analogue) uptake. Rat retina and optic nerve are capable of spontaneous, but temporary, functional recovery after an acute ischemic insult. (C) 2004 Elsevier Ltd. All rights reserved.

Optical imaging of retina in response to grating stimuli in cats

We examined the intrinsic signals in response to grating stimuli in order to determine whether the light-evoked intrinsic signals of the retina are due to changes in the photoreceptor activities induced by the image projected on to the retina or are due to neural activities of the inner retina. The retinas of the left eye of 12 cats under general anesthesia were examined by a functional imaging fundus camera. Near infrared light was used to monitor the reflectance changes (RCs) of the retina. Vertical grating were used to stimulate the retina at 4 Hz. The spatial frequencies of the gratings were 0.05, 0.11, 0.22, 0.43, 0.86, 1.73, and 3.46 cycles/degree (cpd). Ten images were averaged and used to analyze the RCs to obtain the peak value (PV) of a two dimensional fast Fourier transfer of the RCs. The wavefront aberrations (WA) were measured with a compact wavefront aberrometer and the spatial modulation transfer function (MTF) of the eye was calculated. The retinal reflectance image had a grating pattern. The PV of the spatial sensitivity curve was highest at low spatial frequencies (0.05 and 0.11 cpd), and the sensitivity decreased steeply with an increase in the spatial frequency. RCs were not detectable at 3.46 cpd. The MTF decreased gradually with increases in the spatial frequencies and was 0.68 at 3.46 cpd. The reflectance pattern of the retinal intrinsic signal elicited by grating stimuli of different spatial frequencies was different from that of the MTF. This suggests that the intrinsic signal represents not only the response of the photoreceptors but also other neuronal or vascular changes in the retina.

Selenium Distribution in the Human Retina

Selenium is known to occur in the enzyme, glutathione peroxidase, and plays an important role as an antioxidant. The objective of this investigation was to determine if amounts of selenium are selectively accumulated in different regions of the retina or uniformly distributed with eccentricity. 20 human retinas were analyzed for selenium. 18 of these were sectioned into a disc and two concentric annuli centered on the fovea using trephines having diameters of 3, 11, and 21 mm. The sections had areas of7.1, 93, and 343 mm2, respectively. Corresponding sections of these retinas were combined and analyzed together in sets of n = 5 and n = 11. For two donors, the whole retina of one eye was analyzed for selenium and the other retina was sectioned for analysis as described above. Selenium was determined using atomic fluorescence spectroscopy after digestion of the retinal tissues in nitric acid. The two whole retinas were found to have an average of 0.89 ± 0.49 pmoles/mm2 of selenium as compared to the companion which had 0.84 ± 0.28 pmoles/mm2 as determined from the sum of the selenium amounts measured in the individual sections. The inner, medial, and outer portions of these two sectioned retinas were found to contain an average of5.28 ± 1.1, 1.28 ± 0.44, 0.63 ± 0.22 pmoles/mm2, respectively. The five retinas that were sectioned and pooled for analysis were found to have average amounts of3.64, 1.26, and 0.56 pmoles/mm2 • The 11-sectioned retinas were found to have 1.16, 0.61, and 0.38 pmoles/mm2 respectively in the same three sections. This limited data set indicates that selenium is not uniformly distributed within the human retina but rather concentrated to a greater extent within the macula. If confirmed, these data would support the hypothesis that selenium may be an important antioxidant involved in protection of the macula from radical oxidants.

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...

Neural Network Evidence for the Coupling of Presaccadic Visual Remapping to Predictive Eye Position Updating.

As we look around a scene, we perceive it as continuous and stable even though each saccadic eye movement changes the visual input to the retinas. How the brain achieves this perceptual stabilization is unknown, but a major hypothesis is that it relies on presaccadic remapping, a process in which neurons shift their visual sensitivity to a new location in the scene just before each saccade. This hypothesis is difficult to test in vivo because complete, selective inactivation of remapping is currently intractable. We tested it in silico with a hierarchical, sheet-based neural network model of the visual and oculomotor system. The model generated saccadic commands to move a video camera abruptly. Visual input from the camera and internal copies of the saccadic movement commands, or corollary discharge, converged at a map-level simulation of the frontal eye field (FEF), a primate brain area known to receive such inputs. FEF output was combined with eye position signals to yield a suitable coordinate frame for guiding arm movements of a robot. Our operational definition of perceptual stability was "useful stability," quantified as continuously accurate pointing to a visual object despite camera saccades. During training, the emergence of useful stability was correlated tightly with the emergence of presaccadic remapping in the FEF. Remapping depended on corollary discharge but its timing was synchronized to the updating of eye position. When coupled to predictive eye position signals, remapping served to stabilize the target representation for continuously accurate pointing. Graded inactivations of pathways in the model replicated, and helped to interpret, previous in vivo experiments. The results support the hypothesis that visual stability requires presaccadic remapping, provide explanations for the function and timing of remapping, and offer testable hypotheses for in vivo studies. We conclude that remapping allows for seamless coordinate frame transformations and quick actions despite visual afferent lags. With visual remapping in place for behavior, it may be exploited for perceptual continuity.

Development of Swept Source Optical Coherence Tomography and Adaptive Optics Scanning Laser Ophthalmoscopy: Improved Imaging Speed and Handheld Applications

Optical coherence tomography (OCT) is a noninvasive three-dimensional interferometric imaging technique capable of achieving micrometer scale resolution. It is now a standard of care in ophthalmology, where it is used to improve the accuracy of early diagnosis, to better understand the source of pathophysiology, and to monitor disease progression and response to therapy. In particular, retinal imaging has been the most prevalent clinical application of OCT, but researchers and companies alike are developing OCT systems for cardiology, dermatology, dentistry, and many other medical and industrial applications.

Adaptive optics (AO) is a technique used to reduce monochromatic aberrations in optical instruments. It is used in astronomical telescopes, laser communications, high-power lasers, retinal imaging, optical fabrication and microscopy to improve system performance. Scanning laser ophthalmoscopy (SLO) is a noninvasive confocal imaging technique that produces high contrast two-dimensional retinal images. AO is combined with SLO (AOSLO) to compensate for the wavefront distortions caused by the optics of the eye, providing the ability to visualize the living retina with cellular resolution. AOSLO has shown great promise to advance the understanding of the etiology of retinal diseases on a cellular level.

Broadly, we endeavor to enhance the vision outcome of ophthalmic patients through improved diagnostics and personalized therapy. Toward this end, the objective of the work presented herein was the development of advanced techniques for increasing the imaging speed, reducing the form factor, and broadening the versatility of OCT and AOSLO. Despite our focus on applications in ophthalmology, the techniques developed could be applied to other medical and industrial applications. In this dissertation, a technique to quadruple the imaging speed of OCT was developed. This technique was demonstrated by imaging the retinas of healthy human subjects. A handheld, dual depth OCT system was developed. This system enabled sequential imaging of the anterior segment and retina of human eyes. Finally, handheld SLO/OCT systems were developed, culminating in the design of a handheld AOSLO system. This system has the potential to provide cellular level imaging of the human retina, resolving even the most densely packed foveal cones.

Circuits for presaccadic remapping

Saccadic eye movements rapidly displace the image of the world that is projected onto the retinas. In anticipation of each saccade, many neurons in the visual system shift their receptive fields. This presaccadic change in visual sensitivity, known as remapping, was first documented in the parietal cortex and has been studied in many other brain regions. Remapping requires information about upcoming saccades via corollary discharge. Analyses of neurons in a corollary discharge pathway that targets the frontal eye field (FEF) suggest that remapping may be assembled in the FEF’s local microcircuitry. Complementary data from reversible inactivation, neural recording, and modeling studies provide evidence that remapping contributes to transsaccadic continuity of action and perception. Multiple forms of remapping have been reported in the FEF and other brain areas, however, and questions remain about reasons for these differences. In this review of recent progress, we identify three hypotheses that may help to guide further investigations into the structure and function of circuits for remapping.

Circuits for Presaccadic Visual Remapping.

Saccadic eye movements rapidly displace the image of the world that is projected onto the retinas. In anticipation of each saccade, many neurons in the visual system shift their receptive fields. This presaccadic change in visual sensitivity, known as remapping, was first documented in the parietal cortex and has been studied in many other brain regions. Remapping requires information about upcoming saccades via corollary discharge. Analyses of neurons in a corollary discharge pathway that targets the frontal eye field (FEF) suggest that remapping may be assembled in the FEF's local microcircuitry. Complementary data from reversible inactivation, neural recording, and modeling studies provide evidence that remapping contributes to transsaccadic continuity of action and perception. Multiple forms of remapping have been reported in the FEF and other brain areas, however, and questions remain about reasons for these differences. In this review of recent progress, we identify three hypotheses that may help to guide further investigations into the structure and function of circuits for remapping.

Survival or death: a dual role of autophagy in stress-induced pericyte loss in diabetic retinopathy

Aims/hypothesis
Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.
Methods
Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein–LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5 mmol/l) and/or caspase inhibitor Z-VAD-fmk (100 μmol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200 mg protein/l intravitreal concentration) and, after 7 days, retinas were analysed for ER stress, autophagy and apoptosis.
Results
Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50 mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100–200 mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25–200 mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50 mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200 mg/l elicited greater ER stress and apoptosis.
Conclusions
Autophagy has a dual role in diabetic retinopathy: under mild stress (50 mg/l HOG-LDL) it is protective; under more severe stress (200 mg/l HOG-LDL) it promotes cell death.

Diabetes Impairs the Aldehyde Detoxifying Capacity of the Retina

Purpose: We studied whether the accumulation of advanced lipoxidation end-products (ALEs) in the diabetic retina is linked to the impairment of lipid aldehyde detoxification mechanisms.

Methods: Retinas were collected from nondiabetic and diabetic rats and processed for conventional and quantitative RT-PCR (qRT-PCR), Western blotting, immunohistochemistry, and aldehyde dehydrogenase (ALDH) activity assays. The effect of the ALDH1a1 inhibitor, NCT-501, on ALE accumulation and cell viability in cultured Müller glia also was investigated.

Results: The rat retina expressed a range of lipid aldehyde detoxifying ALDH and aldo-keto reductase (AKR) genes. In diabetes, mRNA levels were reduced for 5 of 9 transcripts tested. These findings contrasted with those in the lens and cornea where many of these enzymes were upregulated. We have reported previously accumulation of the acrolein (ACR)-derived ALE, FDP-lysine, in retinal Müller glia during diabetes. In the present study, we show that the main ACR-detoxifying ALDH and AKR genes expressed in the retina, namely, ALDH1a1, ALDH2, and AKR1b1, are principally localized to Müller glia. Diabetes-induced FDP-lysine accumulation in Müller glia was associated with a reduction in ALDH1a1 mRNA and protein expression in whole retina and a decrease in ALDH1a1-immunoreactivity specifically within these cells. No such changes were detected for ALDH2 or AKR1b1. Activity of ALDH was suppressed in the diabetic retina and blockade of ALDH1a1 in cultured Müller glia triggered FDP-lysine accumulation and reduced cell viability.

Conclusions: These findings suggest that downregulation of ALDH and AKR enzymes, particularly ALDH1a1, may contribute ALE accumulation in the diabetic retina.

Development and regeneration of neuronal circuits in the vertebrate retina

Thesis (Ph.D.)--University of Washington, 2016-08

UV wavelengths experienced during development affect larval newt visual sensitivity and predation efficiency

International audience

Factores de riesgo asociados a retinopatía del prematuro en menor o igual a 1750 gramos al nacer, que requirió tratamiento con láser en Hospital de Niños Benjamín Bloom de Enero 2012 a Diciembre 2013

Las retinas inmaduras de los recién nacidos prematuros son susceptibles a insultos que interrumpen el crecimiento neurovascular, que conduce a la retinopatía de la prematuridad. Por eso es de gran importancia conocer los factores de riesgo que se ven involucrados en la génesis de dicha enfermedad. Objetivos: conocer los factores de riesgos asociados a retinopatía del prematuro menor o igual a 1750 gramos al nacer que requirió tratamiento con láser en el Hospital de Niños Benjamín Bloom de Enero de 2012 a Diciembre 2013. Material y método: se realizó un estudio de tipo descriptivo, de corte retrospectivo en el cual se hará revisión sistemática de los expedientes de los pacientes sin dar a conocer la identidad de los mismos, seleccionando a pacientes menor o igual a 1750 gramos independientemente del sexo, evaluado en Hospital Nacional de Niños Benjamín Bloom y al que se le ha realizado intervención con láser; de los cuales se verificará los factores de riesgo asociados al desarrollo de retinopatía del prematuro en estadios severos, se realizó una hoja de datos en Excel y se utilizó el programa SPSS para la agrupación de los datos, posterior se realizó el análisis de los datos. Resultado: se encontraron 56 casos de prematuros con grado severo de retinopatía que utilizó laser, de los cuales fueron 30 masculinos y 26 femeninos; se excluyeron veinte casos por no cumplir criterios de inclusión. Los dos factores de riesgo principalmente involucrados en la ROP son edad gestacional igual o inferior a 30 semanas de gestación y peso al nacimiento igual o inferior a 1300 gr. Otros factores de riesgo encontrados son: la no ganancia de peso para la edad gestacional con un 92.7%, anemia y transfusión sanguínea con un 76.9%, sepsis temprana y nososcomial, en menor proporción apneas, aplicación de surfactante, administración de esteroides prenatales, el estadio con mayor frecuencia encontrado es el III, el déficit visual y ceguera son las secuelas encontradas en los pacientes con retinopatía severa. Conclusiones: la retinopatía de la prematuridad es una entidad multifactorial en la que cada uno de los factores juega un papel importante en la evolución de dicha patología.

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.

Inherited Photoreceptor Degeneration Causes the Death of Melanopsin-Positive Retinal Ganglion Cells and Increases Their Coexpression of Brn3a

Purpose: To study the population of intrinsically photosensitive retinal ganglion cells (melanopsin-expressing RGCs, m+RGCs) in P23H-1 rats, a rat model of inherited photoreceptor degeneration. Methods: At postnatal (P) times P30, P365, and P540, retinas from P23H dystrophic rats (line 1, rapid degeneration; and line 3, slow degeneration) and Sprague Dawley (SD) rats (control) were dissected as whole-mounts and immunodetected for melanopsin and/or Brn3a. The dendritic arborization of m+RGCs and the numbers of Brn3a+RGCs and m+RGCs were quantified and their retinal distribution and coexpression analyzed. Results: In SD rats, aging did not affect the population of Brn3a+RGCs or m+RGCs or the percentage that showed coexpression (0.27%). Young P23H-1 rats had a significantly lower number of Brn3a+RGCs and showed a further decline with age. The population of m+RGCs in young P23H-1 rats was similar to that found in SD rats and decreased by 22.6% and 28.2% at P365 and P540, respectively, similarly to the decrease of the Brn3a+RGCs. At these ages the m+RGCs showed a decrease of their dendritic arborization parameters, which was similar in both the P23H-1 and P23H-3 lines. The percentage of coexpression of Brn3a was, however, already significantly higher at P30 (3.31%) and increased significantly with age (10.65% at P540). Conclusions: Inherited photoreceptor degeneration was followed by secondary loss of Brn3a+RGCs and m+RGCs. Surviving m+RGCs showed decreased dendritic arborization parameters and increased coexpression of Brn3a and melanopsin, phenotypic and molecular changes that may represent an effort to resist degeneration and/or preferential survival of m+RGCs capable of synthesizing Brn3a.