Melanopsin+RGCs Are fully Resistant to NMDA-Induced Excitotoxicity

We studied short- and long-term effects of intravitreal injection of N-methyl-d-aspartate (NMDA) on melanopsin-containing (m+) and non-melanopsin-containing (Brn3a+) retinal ganglion cells (RGCs). In adult SD-rats, the left eye received a single intravitreal injection of 5µL of 100nM NMDA. At 3 and 15 months, retinal thickness was measured in vivo using Spectral Domain-Optical Coherence Tomography (SD-OCT). Ex vivo analyses were done at 3, 7, or 14 days or 15 months after damage. Whole-mounted retinas were immunolabelled for brain-specific homeobox/POU domain protein 3A (Brn3a) and melanopsin (m), the total number of Brn3a+RGCs and m+RGCs were quantified, and their topography represented. In control retinas, the mean total numbers of Brn3a+RGCs and m+RGCs were 78,903 ± 3572 and 2358 ± 144 (mean ± SD; n = 10), respectively. In the NMDA injected retinas, Brn3a+RGCs numbers diminished to 49%, 28%, 24%, and 19%, at 3, 7, 14 days, and 15 months, respectively. There was no further loss between 7 days and 15 months. The number of immunoidentified m+RGCs decreased significantly at 3 days, recovered between 3 and 7 days, and were back to normal thereafter. OCT measurements revealed a significant thinning of the left retinas at 3 and 15 months. Intravitreal injections of NMDA induced within a week a rapid loss of 72% of Brn3a+RGCs, a transient downregulation of melanopsin expression (but not m+RGC death), and a thinning of the inner retinal layers.This study was supported by the Fundación Séneca, Agencia de Ciencia y Tecnología Región de Murcia (19881/GERM/15), and the Spanish Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, Fondo Europeo de Desarrollo Regional “una manera de hacer Europa” (SAF2015-67643-P, PI16/00380, RD16/0008/0026 and RD16/0008/0016)

Retinal Molecular Changes Are Associated with Neuroinflammation and Loss of RGCs in an Experimental Model of Glaucoma

Signaling mediated by cytokines and chemokines is involved in glaucoma-associated neuroinflammation and in the damage of retinal ganglion cells (RGCs). Using multiplexed immunoassay and immunohistochemical techniques in a glaucoma mouse model at different time points after ocular hypertension (OHT), we analyzed (i) the expression of pro-inflammatory cytokines, anti-inflammatory cytokines, BDNF, VEGF, and fractalkine; and (ii) the number of Brn3a+ RGCs. In OHT eyes, there was an upregulation of (i) IFN-γ at days 3, 5, and 15; (ii) IL-4 at days 1, 3, 5, and 7 and IL-10 at days 3 and 5 (coinciding with downregulation of IL1-β at days 1, 5, and 7); (iii) IL-6 at days 1, 3, and 5; (iv) fractalkine and VEGF at day 1; and (v) BDNF at days 1, 3, 7, and 15. In contralateral eyes, there were (i) an upregulation of IL-1β at days 1 and 3 and a downregulation at day 7, coinciding with the downregulation of IL4 at days 3 and 5 and the upregulation at day 7; (ii) an upregulation of IL-6 at days 1, 5, and 7 and a downregulation at 15 days; (iii) an upregulation of IL-10 at days 3 and 7; and (iv) an upregulation of IL-17 at day 15. In OHT eyes, there was a reduction in the Brn3a+ RGCs number at days 3, 5, 7, and 15. OHT changes cytokine levels in both OHT and contralateral eyes at different time points after OHT induction, confirming the immune system involvement in glaucomatous neurodegeneration

Animal Models of LED-Induced Phototoxicity. Short- and Long-Term In Vivo and Ex Vivo Retinal Alterations

Phototoxicity animal models have been largely studied due to their degenerative communalities with human pathologies, e.g., age-related macular degeneration (AMD). Studies have documented not only the effects of white light exposure, but also other wavelengths using LEDs, such as blue or green light. Recently, a blue LED-induced phototoxicity (LIP) model has been developed that causes focal damage in the outer layers of the superior-temporal region of the retina in rodents. In vivo studies described a progressive reduction in retinal thickness that affected the most extensively the photoreceptor layer. Functionally, a transient reduction in a- and b-wave amplitude of the ERG response was observed. Ex vivo studies showed a progressive reduction of cones and an involvement of retinal pigment epithelium cells in the area of the lesion and, in parallel, an activation of microglial cells that perfectly circumscribe the damage in the outer retinal layer. The use of neuroprotective strategies such as intravitreal administration of trophic factors, e.g., basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) or pigment epithelium-derived factor (PEDF) and topical administration of the selective alpha-2 agonist (Brimonidine) have demonstrated to increase the survival of the cone population after LIP

Short- and Long-Term Study of the Impact of Focal Blue Light-Emitting Diode-Induced Phototoxicity in Adult Albino Rats

Background: In adult rats we study the short- and long-term effects of focal blue light-emitting diode (LED)-induced phototoxicity (LIP) on retinal thickness and Iba-1+ activation. Methods: The left eyes of previously dark-adapted Sprague Dawley (SD) rats were photoexposed to a blue LED (20 s, 200 lux). In vivo longitudinal monitoring of retinal thickness, fundus images, and optical retinal sections was performed from 1 to 30 days (d) after LIP with SD-OCT. Ex vivo, we analysed the population of S-cone and Iba-1+ cells within a predetermined fixed-size circular area (PCA) centred on the lesion. Results: LIP resulted in a circular focal lesion readily identifiable in vivo by fundus examination, which showed within the PCAs a progressive thinning of the outer retinal layer, and a diminution of the S-cone population to 19% by 30 d. In parallel to S-cone loss, activated Iba-1+ cells delineated the lesioned area and acquired an ameboid morphology with peak expression at 3 d after LIP. Iba-1+ cells adopted a more relaxed-branched morphology at 7 d and by 14–30 d their morphology was fully branched. Conclusion: LIP caused a progressive reduction of the outer retina with loss of S cones and a parallel dynamic activation of microglial cells in the lesioned area

Pigment Epithelium-Derived Factor (PEDF) Fragments Prevent Mouse Cone Photoreceptor Cell Loss Induced by Focal Phototoxicity In Vivo

Here, we evaluated the effects of PEDF (pigment epithelium-derived factor) and PEDF peptides on cone-photoreceptor cell damage in a mouse model of focal LED-induced phototoxicity (LIP) in vivo. Swiss mice were dark-adapted overnight, anesthetized, and their left eyes were exposed to a blue LED placed over the cornea. Immediately after, intravitreal injection of PEDF, PEDF-peptide fragments 17-mer, 17-mer[H105A] or 17-mer[R99A] (all at 10 pmol) were administered into the left eye of each animal. BDNF (92 pmol) and bFGF (27 pmol) injections were positive controls, and vehicle negative control. After 7 days, LIP resulted in a consistent circular lesion located in the supratemporal quadrant and the number of S-cones were counted within an area centered on the lesion. Retinas treated with effectors had significantly greater S-cone numbers (PEDF (60%), 17-mer (56%), 17-mer [H105A] (57%), BDNF (64%) or bFGF (60%)) relative to their corresponding vehicle groups (≈42%). The 17-mer[R99A] with no PEDF receptor binding and no neurotrophic activity, PEDF combined with a molar excess of the PEDF receptor blocker P1 peptide, or with a PEDF-R enzymatic inhibitor had undetectable effects in S-cone survival. The findings demonstrated that the cone survival effects were mediated via interactions between the 17-mer region of the PEDF molecule and its PEDF-R receptor