We have investigated the effects of light-emitting diode (LED)-induced phototoxicity (LIP) on cone-photoreceptors and their protection with brimonidine (BMD), brain-derived neurotrophic factor ...(BDNF), pigment epithelium-derived factor (PEDF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF). In anesthetized, dark adapted, adult albino rats a blue (400 nm) LED was placed perpendicular to the cornea (10 sec, 200 lux) and the effects were investigated using Spectral Domain Optical Coherence Tomography (SD-OCT) and/or analysing the retina in oriented cross-sections or wholemounts immune-labelled for L- and S-opsin and counterstained with the nuclear stain DAPI. The effects of topical BMD (1%) or, intravitreally injected BDNF (5 µg), PEDF (2 µg), CNTF (0.4 µg) or bFGF (1 µg) after LIP were examined on wholemounts at 7 days. SD-OCT showed damage in a circular region of the superotemporal retina, whose diameter varied from 1,842.4±84.5 µm (at 24 hours) to 1,407.7±52.8 µm (at 7 days). This region had a progressive thickness diminution from 183.4±5 µm (at 12 h) to 114.6±6 µm (at 7 d). Oriented cross-sections showed within the light-damaged region of the retina massive loss of rods and cone-photoreceptors. Wholemounts documented a circular region containing lower numbers of L- and S-cones. Within a circular area (1 mm or 1.3 mm radius, respectively) in the left and in its corresponding region of the contralateral-fellow-retina, total L- or S-cones were 7,118±842 or 661±125 for the LED exposed retinas (n = 7) and 14,040±1,860 or 2,255±193 for the fellow retinas (n = 7), respectively. BMD, BDNF, PEDF and bFGF but not CNTF showed significant neuroprotective effects on L- or S-cones. We conclude that LIP results in rod and cone-photoreceptor loss, and is a reliable, quantifiable model to study cone-photoreceptor degeneration. Intravitreal BDNF, PEDF or bFGF, or topical BMD afford significant cone neuroprotection in this model.
To identify and characterize numerically and topographically the population of alpha retinal ganglion cells (αRGCs) and their subtypes, the sustained-response ON-center αRGCs (ONs-αRGCs), which ...correspond to the type 4 intrinsically photosensitive RGCs (M4-ipRGCs), the transient-response ON-center αRGCs (ONt-αRGCs), the sustained-response OFF-center αRGCs (OFFs-αRGCs), and the transient-response OFF-center αRGCs (OFFt-αRGCs) in the adult pigmented mouse retina.
The αRGC population and its subtypes were studied in flat-mounted retinas and radial sections immunodetected against non-phosphorylated high molecular weight neurofilament subunit (SMI-32) or osteopontin (OPN), two αRGCs pan-markers; Calbindin, expressed in ONs-αRGCs, and amacrines; T-box transcription factor T-brain 2 (Tbr2), a key transcriptional regulator for ipRGC development and maintenance, expressed in ipRGCs and GABA-displaced amacrine cells; OPN4, an anti-melanopsin antibody; or Brn3a and Brn3c, markers of RGCs. The total population of RGCs was counted automatically and αRGCs and its subtypes were counted manually, and color-coded neighborhood maps were used for their topographical representation.
The total mean number of αRGCs per retina is 2,252 ± 306 SMI32
αRGCs and 2,315 ± 175 OPN
αRGCs (
= 10), representing 5.08% and 5.22% of the total number of RGCs traced from the optic nerve, respectively. αRGCs are distributed throughout the retina, showing a higher density in the temporal hemiretina. ONs-αRGCs represent ≈36% 841 ± 110 cells (
= 10) of all αRGCs and are located throughout the retina, with the highest density in the temporal region. ONt-αRGCs represent ≈34% 797 ± 146 cells (
= 10) of all αRGCs and are mainly located in the central retinal region. OFF-αRGCs represent the remaining 32% of total αRGCs and are divided equally between OFFs-αRGCs and OFFt-αRGCs 363 ± 50 cells (
= 10) and 376 ± 36 cells (
= 10), respectively. OFFs-αRGCs are mainly located in the supero-temporal peripheral region of the retina and OFFt-αRGCs in the mid-peripheral region of the retina, especially in the infero-temporal region.
The combination of specific antibodies is a useful tool to identify and study αRGCs and their subtypes. αRGCs are distributed throughout the retina presenting higher density in the temporal area. The sustained ON and OFF response subtypes are mainly located in the periphery while the transient ON and OFF response subtypes are found in the central regions of the retina.
Purpose: To study the microglial reaction and degeneration of the retinal pigment epithelium (RPE) and its protection with basic fibroblast growth factor (bFGF) administered alone or combined with ...minocycline (MC) in a model of focal light‐emitting diode (LED)‐induced phototoxicity (LIP) in mice.
Methods: In dark‐adapted adult C57 mice (20–25 g), the left eye was dilated and exposed for 45 seconds to a blue LED (400 nm; 500 lux) placed 2 mm perpendicular to the corneal apex (n = 71). Mice were treated with bFGF (0.5 μg) administered alone or in combination with MC (45 mg/kg). bFGF was administered in a single intravitreal injection just after LIP and the MC was daily injected intraperitoneally, starting the day before LIP (n = 10–13 per group). Vehicle or naïve groups were used as controls (n = 6–10 each group). Retinas were immunodetected with anti‐arrestin and anti‐Iba1 antibodies to study cone outer segments (a+OS) and microglial cells (Iba1+cells) and RPEs were immunodetected with anti‐ZO1 to study RPE cells, at 3 and 7 days after LIP. The number of cones and microglial cells was studied within a pre‐fixed area (PFA) of 0.9 mm diameter at the centre of the lesion.
Results: Treatment with bFGF alone or in combination with MC was effective in protecting a+OS within the PFA compared to vehicles at 7 days after LIP (p = 0.034 and p = 0.001, respectively). The combined treatment of bFGF and MC was effective in reducing the number of Iba1+cells within the PFA in the outer plexiform layer at 7 days after LIP (p = 0.0003). However, the treatments had no restorative effect on RPE cell survival or morphological integrity at 7 days after LIP (p = 0.6762).
Conclusions: Administration of bFGF alone or in combination with MC increased a+OS survival and reduces Iba1+cell activation but does not reduce RPE damage.
Purpose: To study the effect of minocycline administration on the evolution of retinal ganglion cell (RGC) loss, the number of microglial cells and the activation of the apoptotic signal Caspase‐3 ...after ocular hypertension (OHT) in mice.
Methods: In left eyes of Swiss albino mice, OHT was induced by diode laser photocoagulation of the limbal and episcleral veins and the intraocular pressure (IOP) evolution was monitored with a TonoLab®. Mice received daily intraperitoneal injections of minocycline hydrochloride (45 mg/kg) diluted in saline starting the day before OHT induction until the day of sacrifice (4 or 15 days, n = 12–14 per group/time point). Control groups received saline injections. Retinas were divided into two groups. Retinas of the first group were double immunodetected against Brn3a and active Caspase‐3 to identify surviving (Brn3a+RGCs) and apoptotic (a‐Casp3+RGCs) RGCs. In the second group, RGCs were retrogradely traced with Fluorogold 7 days before OHT induction and retinas were immunodetected against Iba‐1. Total Brn3a+RGCs, a‐Casp3+RGCs, Iba‐1+cells and FG+microglial cells in the RGC layer were manually or automatically quantified, and the distributions were analysed by topographical maps.
Results: IOP was significantly elevated 24 hours after OHT induction (33.2 ± 6.1 mmHg) and returned to baseline at 1 week (17.3 ± 5.3 mmHg). This evolution was not altered by minocycline administration. Although minocycline treatment was not successful in protecting Brn3a+RGCs at 4 (40 878 ± 4860 vs 43 526 ± 2714) nor at 15 (16 161 ± 12 650 vs 12 919 ± 6085) days after OHT, the numbers of a‐Casp3+RGCs, Iba‐1+cells and FG+microglial cells were significantly lower in the minocycline‐treated groups at both 4 (58.6%, 46.8% and 24.5%, respectively) and 15 (33.6%, 54.2% and 37.3%, respectively) days compared to the saline‐treated groups (100%).
Conclusions: Minocycline treatment decreases the number of microglial cells and caspase‐3 activation but does not reduce RGC loss after OHT.
Purpose: To study the role of taurine in the morphology and phagocytic function of the retinal pigment epithelium (RPE) in normal and dystrophic rats.
Methods: Albino Sprague–Dawley (SD) rats and ...pigmented dystrophic Royal College of Surgeons (RCS‐p+) rats that suffer a severe form of inherited photoreceptor degeneration due to RPE phagocytosis impairment, were used for this study. SD rats received β‐alanine in drinking water (3%) for 2 months from P21 to induce taurine depletion and RCS‐p + rats received taurine supplementation in drinking water (0.2 M) for 24 days from P21. Groups of untreated RCS and SD rats were used as control. SD rats were processed 2 months after the start of treatment, while RCS rats were processed at P45. Twenty‐four hours before processing, the animals received an intravitreal injection of 1.5 μl of 3% fluorogold (FG) diluted in saline 24 to label the RPE cells. Animals were processed and the RPE was dissected flat‐mounted and observed using a confocal microscope (Leica SP8).
Results: In control SD animals, Fluorogold (FG) accumulates homogeneously in the cytoplasm of the RPE cells. Taurine‐depleted SD animals showed less FG accumulation in the cytoplasm in all cells and, and some cells had FG accumulation only in part of the cytoplasm or no FG accumulation at all. Untreated RCS‐p + animals showed an inverted accumulation of FG because it appeared mainly in the membrane of the RPE but not in the cytoplasm. Finally, taurine‐treated RCS‐p + animals showed FG accumulation in the cytoplasm of the RPE cells, although the accumulation of FG was smaller than that found in SD rats.
Conclusions: Taurine influences the phagocytic capacity of the RPE cells. Taurine depletion impairs the phagocytic capacity of the RPE cells in healthy retinas, and taurine supplementation improves the phagocytic function of RPE cells in dystrophic RCS‐p + rats.
To analyze the role of microglial and Müller cells in the formation of rings of photoreceptor degeneration caused by phototoxicity.
Two-month-old Sprague-Dawley rats were exposed to light and ...processed 1, 2, or 3 months later. Retinas were dissected as whole-mounts, immunodetected for microglial cells, Müller cells, and S- and L/M-cones and analyzed using fluorescence, thunder imaging, and confocal microscopy. Cone populations were automatically counted and isodensity maps constructed to document cone topography.
Phototoxicity causes a significant progressive loss of S- and L/M-cones of up to 68% and 44%, respectively, at 3 months after light exposure (ALE). One month ALE, we observed rings of cone degeneration in the photosensitive area of the superior retina. Two and 3 months ALE, these rings had extended to the central and inferior retina. Within the rings of cone degeneration, there were degenerating cones, often activated microglial cells, and numerous radially oriented processes of Müller cells that showed increased expression of intermediate filaments. Between 1 and 3 months ALE, the rings coalesced, and at the same time the microglial cells resumed a mosaic-like distribution, and there was a decrease of Müller cell gliosis at the areas devoid of cones.
Light-induced photoreceptor degeneration proceeds with rings of cone degeneration, as observed in inherited retinal degenerations in which cone death is secondary to rod degeneration. The spatiotemporal relationship of cone death microglial cell activation and Müller cell gliosis within the rings of cone degeneration suggests that, although both glial cells are involved in the formation of the rings, they may have coordinated actions and, while microglial cells may be more involved in photoreceptor phagocytosis, Müller cells may be more involved in cone and microglial cell migration, retinal remodeling and glial seal formation.
Purpose
To develop a new model of focal Light Emitting Diode‐Induced photoreceptor degeneration in adult pigmented mice.
Methods
In adult female C57 mice (20 g) dark adapted, the left eye was dilated ...with clycopentolate. Mice were anesthetized and exposed during 45 seconds to a blue (400 nm) light emitting diode (LED) (500 lux) placed 2 mm in front of the corneal apex. Animals were sacrificed 1, 3, 5 or 7 days (n = 6 each) after LED exposure, their retinas were dissected, prepared as whole‐mounts, doubly immunolabeled with antibodies against arrestin and Iba‐1 to identify cones and microglial cells and examined under fluorescence and confocal microscopy. Spectral Domain Optical Coherence Tomography (SD‐OCT, Spectralis, Heidelberg) was used to examine longitudinally in vivo the retinal thickness in both eyes at the different intervals after LED exposure.
Results
SD‐OCT eye fundus imaging with infrared reflectance allowed to document in vivo in the supero‐temporal quadrant of the LED exposed retina a rounded less reflective area that showed progressive thinning of the outer retina (approximately 75%) from 1–7 days after LED exposure. This light‐sensitive area showed by 1 day shortened cone outer segments, by 3 days a clear diminution of cone density, and by 7 days additional cone loss. The outer layers of the retina in the light‐sensitive area showed 1 day after LED exposure Iba1+ cells in the center that remained in the same location for 5–7 days. The morphology of these Iba1+ cells was dendritic at 1 day, became ameboid at 3 days and dendritic again at 5–7 days.
Conclusions
LED can be used to produce a focal phototoxic lesion in the retina. The focal lesion shows decreased retinal thickness, cone densities and an activation of microglial cells in the outer retinal layers.
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