Marcelino Avilés-Trigueros

Emerging evidence suggests that retinal neurodegeneration is an early event in the pathogenesis of diabetic retinopathy (DR), preceding the development of microvascular abnormalities. Here, we assessed the impact of neuroinflammation on the retina of diabetic-induced rats. For this aim we have used a two-photon microscope to image the photoreceptors (PRs) at different eccentricities in unstained retinas obtained from both control (N = 4) and pathological rats (N = 4). This technique provides high-resolution images where individual PRs can be identified. Within each image, every PR was located, and its transversal area was measured and used as an objective parameter of neuroinflammation. In control samples, the size of the PRs hardly changed with retinal eccentricity. On the opposite end, diabetic retinas presented larger PR transversal sections. The ratio of PRs suffering from neuroinflammation was not uniform across the retina. Moreover, the maximum anatomical resolving power (in cycles/deg) was also calculated. This presents a double-slope pattern (from the central retina towards the periphery) in both types of specimens, although the values for diabetic retinas were significantly lower across all retinal locations. The results show that chronic retinal inflammation due to diabetes leads to an increase in PR transversal size. These changes are not uniform and depend on the retinal location. Two-photon microscopy is a useful tool to accurately characterize and quantify PR inflammatory processes and retinal alterations.

Purpose: The aim of this study was to investigate, the neuroprotective effects of a new Gramine derivative named: ITH12657, in a model of retinal excitotoxicity induced by intravitreal injection of NMDA.Methods: Adult Sprague Dawley rats received an intravitreal injection of 100 mM NMDA in their left eye and were treated daily with subcutaneous injections of ITH12657 or vehicle. The best dose–response, therapeutic window study, and optimal treatment duration of ITH12657 were studied. Based on the best survival of Brn3a + RGCs obtained from the above-mentioned studies, the protective effects of ITH12657 were studied in vivo (retinal thickness and full-field Electroretinography), and ex vivo by quantifying the surviving population of Brn3a + RGCs, αRGCs and their subtypes α-ONsRGCs, α-ONtRGCs, and α-OFFRGCs.Results: Administration of 10 mg/kg ITH12657, starting 12 h before NMDA injection and dispensed for 3 days, resulted in the best significant protection of Brn3a + RGCs against NMDA-induced excitotoxicity. In vivo, ITH12657-treated rats showed significant preservation of retinal thickness and functional protection against NMDA-induced retinal excitotoxicity. Ex vivo results showed that ITH12657 afforded a significant protection against NMDA-induced excitotoxicity for the populations of Brn3a + RGC, αRGC, and αONs-RGC, but not for the population of αOFF-RGC, while the population of α-ONtRGC was fully resistant to NMDA-induced excitotoxicity.Conclusion: Subcutaneous administration of ITH12657 at 10 mg/kg, initiated 12 h before NMDA-induced retinal injury and continued for 3 days, resulted in the best protection of Brn3a + RGCs, αRGC, and αONs-RGC against excitotoxicity-induced RGC death. The population of αOFF-RGCs was extremely sensitive while α-ONtRGCs were fully resistant to NMDA-induced excitotoxicity.

To analyze the neuroprotective effects of 7,8-Dihydroxyflavone (DHF) in vivo and ex vivo, adult albino Sprague-Dawley rats were given a left intraorbital optic nerve transection (IONT) and were divided in two groups: One was treated daily with intraperitoneal (ip) DHF (5 mg/kg) (n = 24) and the other (n = 18) received ip vehicle (1% DMSO in 0.9% NaCl) from one day before IONT until processing. At 5, 7, 10, 12, 14, and 21 days (d) after IONT, full field electroretinograms (ERG) were recorded from both experimental and one additional naïve-control group (n = 6). Treated rats were analyzed 7 (n = 14), 14 (n = 14) or 21 d (n = 14) after IONT, and the retinas immune stained against Brn3a, Osteopontin (OPN) and the T-box transcription factor T-brain 2 (Tbr2) to identify surviving retinal ganglion cells (RGCs) (Brn3a⁺), α-like (OPN⁺), α-OFF like (OPN⁺Brn3a⁺) or M4-like/α-ON sustained RGCs (OPN⁺Tbr⁺). Naïve and right treated retinas showed normal ERG recordings. Left vehicle-treated retinas showed decreased amplitudes of the scotopic threshold response (pSTR) (as early as 5 d), the rod b-wave, the mixed response and the cone response (as early as 10 d), which did not recover with time. In these retinas, by day 7 the total numbers of Brn3a⁺RGCs, OPN⁺RGCs and OPN⁺Tbr2⁺RGCs decreased to less than one half and OPN⁺Brn3a⁺RGCs decreased to approximately 0.5%, and Brn3a⁺RGCs showed a progressive loss with time, while OPN⁺RGCs and OPN⁺Tbr2⁺RGCs did not diminish after seven days. Compared to vehicle-treated, the left DHF-treated retinas showed significantly greater amplitudes of the pSTR, normal b-wave values and significantly greater numbers of OPN⁺RGCs and OPN⁺Tbr2⁺RGCs for up to 14 d and of Brn3a⁺RGCs for up to 21 days. DHF affords significant rescue of Brn3a⁺RGCs, OPN⁺RGCs and OPN⁺Tbr2⁺RGCs, but not OPN⁺Brn3a⁺RGCs, and preserves functional ERG responses after IONT.

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.

Purpose: To analyze microglial cells activation features, and P2RY12, MHC‐II and CD68 expression in 15‐month‐old mice compared to young adult mice, after unilateral laser‐induced ocular hypertension (OHT)Methods: Albino Swiss mice divided in four groups; young naïve (n = 6), aged naïve (n = 6), young OHT (YG) (n = 6) and aged OHT (AG) (n = 6), were analyzed. In OHT groups, both OHT eyes and contralateral eyes were studied. Retinal whole‐mounts were processed with: 1) anti Iba‐1 to quantify: i) Iba‐1 + cells number (Ibacn) in outer segments (OS), outer plexiform layer (OPL) and inner plexiform layer (IPL); ii) area of retina occupied by Iba‐1 + cells (Iba‐1 RA) in the nerve fiber layer‐ ganglion cell layer (NFL‐GCL) iii) cell body area of Iba‐1+ cells (CbIbac) in OPL, IPL and NFL‐GCL; iv) arbor area of Iba‐1+ cells (AAIbac) in OPL and IPL; and vi) vertical processes number of Iba‐1+ cells (VPIbac) between OS and OPL. 2) anti‐P2RY12, (resident microglia), anti‐CD68 (phagocytic activity) and anti‐MHC‐II (microglial activation marker)Results: Both OHT eyes and contralateral eyes of YG and AG showed significant: increase of Ibacn, Iba‐1 RA, CbIbac and VPIbac and decrease of AAIbac, more pronounced in OHT eyes, compared to their respective naïve groups. Comparing YG and AG showed: i) AG versus YG in OHT eyes, significant decrease of Iba‐1 RA and VPIbac; ii) AG versus YG in contralateral eyes, a significant increase of Ibacn (OS) and a significant decrease of CbIbac and AAIbac (IPL). Analyzing the P2RY12, CD68 and MHC‐II expression we observed: i) in YG all Iba‐1+ cells were P2RY12+, except perivascular and dendritic cells, but in AG most cells were Iba‐1+/P2RY12‐; ii) in AG and YG numerous amoeboid‐like CD68+ cells were found but in AG ramified appearance CD68+ cells were also observed, and iii) While in YG practically all Iba‐1+ cells were MHC‐II+, in AG some Iba‐1+ cells showed MHC‐II+ expressionConclusions: The morphological and molecular differences found in microglial cells of AG compared to YG show the possible aging implication in glaucoma.

Purpose: To quantify different morphological signs of microglial cells activation, as well as to analyze the P2RY12, MHC‐II and CD68 expression in 15‐month old mice , an early stage of aging (aged naïve) in comparison with a young adult mice (young naïve).Methods: Young naïve (n = 6) and aged naïve (n = 6) mice were analyzed. Retinal whole‐mounts were immunolabeled with: 1) anti Iba‐1 to quantify: i) Iba‐1 + cells number (Ibacn) in outer segments (OS), outer plexiform layer (OPL) and inner plexiform layer (IPL); ii) area of retina occupied by Iba‐1 + cells (Iba‐1 RA) in the nerve fiber layer‐ ganglion cell layer (NFL‐GCL) iii) cell body area of Iba‐1+ cells (CbIbac) in OPL, IPL and NFL‐GCL; iv) arbor area of Iba‐1+ cells (AAIbac) in OPL and IPL; number of vertical processes of Iba‐1+ cells (VPIbac) beetween the OS and OPL. 2) anti‐P2RY12 to identify resident microglia, anti‐CD68 and anti‐MHC‐II as a microglial activation marker.Results: In aged naïve with respect to young naïve the quantitative analysis showed: i) a non‐significant increase in the Ibacn in OS; ii) a non‐significant decrease in the AAIbac in OPL; iii) a significant increase in the CbIbac in OPL, IPL and NFL‐GCL and iv) a significant increase in the VPIbac. When we analyzed the expression of P2RY12, CD68 and MHC‐II we observed: i) in young naïve all Iba‐1+ cells were P2RY12+, except perivascular and dendritic cells, but in the aged naïve some cells were Iba‐1+/P2RY12‐; ii) in aged naïve numerous amoeboid‐like CD68+ cells were found while in the young naïve the expression of CD68 practically was absent, and iii) in both young naïve and aged naïve no expression of MHC‐II was observed.Conclusions: In 15‐month‐old mice, morphological changes and in the expression of P2RY12, CD68 and MHC‐II occurs in the microglial cells compared to young adult mice. These signs show a non‐pathological state of microglial activation that could influence of retinal age‐related disorders.

Background: To induce a moderate chronic ocular hypertension (OHT) by injecting polidocanol, a foamed sclerosant drug, in the aqueous humor outflow pathway. Methods: Intraocular pressure (IOP) was monitored for up to 6 months. Pattern and full-field electroretinogram (PERG and ERG) were recorded and retinal ganglion cells (RGC) and retinal nerve fiber layer (RNFL) thickness were assessed in vivo with optical coherence tomography (OCT) and ex vivo using Brn3a immunohistochemistry. Results: In the first 3 weeks post-injection, a significant IOP elevation was observed in the treated eyes (18.47 ± 3.36 mmHg) when compared with the control fellow eyes (12.52 ± 2.84 mmHg) (p < 0.05). At 8 weeks, 65% (11/17) of intervention eyes had developed an IOP increase >25% over the baseline. PERG responses were seen to be significantly reduced in the hypertensive eyes (2.25 ± 0.24 µV) compared to control eyes (1.44 ± 0.19 µV) (p < 0.01) at week 3, whereas the ERG components (photoreceptor a-wave and bipolar cell b-wave) remained unaltered. By week 24, RNFL thinning and cell loss in the ganglion cell layer was first detected (2/13, 15.3%) as assessed by OCT and light microscopy. Conclusions: This novel OHT rat model, with moderate levels of chronically elevated IOP, and abnormal PERG shows selective functional impairment of RGC.