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Abstract Background Ocular hypertension is a major risk factor for glaucoma, a neurodegenerative disease characterized by an irreversible decrease in ganglion cells and their axons. Macroglial and microglial cells appear to play an important role in the pathogenic mechanisms of the disease. Here, we study the effects of laser-induced ocular hypertension (OHT) in the macroglia, microglia and retinal ganglion cells (RGCs) of eyes with OHT (OHT-eyes) and contralateral eyes two weeks after lasering. Methods Two groups of adult Swiss mice were used: age-matched control (naïve, n = 9); and lasered (n = 9). In the lasered animals, both OHT-eyes and contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against glial fibrillary acid protein (GFAP), neurofilament of 200kD (NF-200), ionized calcium binding adaptor molecule (Iba-1) and major histocompatibility complex class II molecule (MHC-II). The GFAP-labeled retinal area (GFAP-RA), the intensity of GFAP immunoreaction (GFAP-IR), and the number of astrocytes and NF-200 + RGCs were quantified. Results In comparison with naïve: i) astrocytes were more robust in contralateral eyes. In OHT-eyes, the astrocyte population was not homogeneous, given that astrocytes displaying only primary processes coexisted with astrocytes in which primary and secondary processes could be recognized, the former having less intense GFAP-IR (P P P P P = 0.05) and in OHT-eyes (P Conclusion The use of the contralateral eye as an internal control in experimental induction of unilateral IOP should be reconsidered. The gliotic behavior in contralateral eyes could be related to the immune response. The absence of NF-200+RGCs (sign of RGC degeneration) leads us to postulate that the MHC-II upregulation in contralateral eyes could favor neuroprotection.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Di Pierdomenico, Gallego-Ortega, Norte-Muñoz, Vidal-Villegas, Bravo, Boluda-Ruiz, Bernal-Garro, Fernandez-Bueno, Pastor-Jimeno, Villegas-Pérez, Avilés-Trigueros, de los Ríos and Vidal-Sanz.)

Purpose: 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. Methods: 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. Results: The total mean number of αRGCs per retina is 2,252 ± 306 SMI32 + αRGCs and 2,315 ± 175 OPN + αRGCs ( n = 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 ( n = 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 ( n = 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 ( n = 10) and 376 ± 36 cells ( n = 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. Conclusions: 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gallego-Ortega, Norte-Muñoz, Di Pierdomenico, Avilés-Trigueros, de la Villa, Valiente-Soriano and Vidal-Sanz.)

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.

Diabetic retinopathy (DR) is defined as a microvascular pathology. However, some data have suggested that the retinal photoreceptors (PRs) might be important in the pathogenesis of this ocular disease. In this study the organization of the PRs in control and diabetic-induced rats was compared using multiphoton microscopy. The PR mosaic was imaged at different locations in non-stained retinas. The density of PRs was directly quantified from cell counting. The spatially resolved density presents a double-slope pattern (from the central retina towards the periphery) in both healthy and pathological samples, although the values for the latter were significantly lower all across the retina. Moreover, Voronoi analysis was performed to explore changes in PR topography. In control specimens a hexagonally packed structure was dominant. However, despite the non-controlled effects of the disease in retinal structures, this PR regularity was fairly maintained in diabetic retinas., Competing Interests: The authors declare no conflicts of interest., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Microglial activation is associated with glaucoma. In the model of unilateral laser-induced ocular hypertension (OHT), the time point at which the inflammatory process peaks remains unknown. Different time points (1, 3, 5, 8, and 15 d) were compared to analyze signs of microglial activation both in OHT and contralateral eyes. In both eyes, microglial activation was detected in all retinal layers at all time points analyzed, including: i) increase in the cell number in the outer segment photoreceptor layer and plexiform layers (only in OHT eyes) from 3 d onward; ii) increase in soma size from 1 d onward; iii) retraction of the processes from 1 d in OHT eyes and 3 d in contralateral eyes; iv) increase in the area of the retina occupied by Iba-1+ cells in the nerve fiber layer/ganglion cell layer from 1 d onward; v) increase in the number of vertical processes from 1 d in contralateral eyes and 3 d in OHT eyes. In OHT eyes at 24 h and 15 d, most Iba-1+ cells were P2RY12+ and were down-regulated at 3 and 5 d. In both eyes, microglial activation was stronger at 3 and 5 d (inflammation peaked in this model). These time points could be useful to identify factors implicated in the inflammatory process.

To study short and long-term effects of acute ocular hypertension (AOHT) on inner and outer retinal layers, in adult Sprague-Dawley rats AOHT (87mmHg) was induced for 90min and the retinas were examined longitudinally in vivo with electroretinogram (ERG) recordings and optical coherent tomography (OCT) from 1 to 90 days (d). Ex vivo, the retinas were analyzed for rod (RBC) and cone (CBC) bipolar cells, with antibodies against protein kinase Cα and recoverin, respectively in cross sections, and for cones, horizontal (HZ) and ganglion (RGC) cells with antibodies against arrestin, calbindin and Brn3a, respectively in wholemounts. The inner retina thinned progressively up to 7d with no further changes, while the external retina had a normal thickness until 30d, with a 20% thinning between 30 and 90d. Functionally, the a-wave showed an initial reduction by 24h and a further reduction from 30 to 90d. All other main ERG waves were significantly reduced by 1d without significant recovery by 90d. Radial sections showed a normal population of RBCs but their terminals were reduced. The CBCs showed a progressive decrease with a loss of 56% by 30d. In wholemount retinas, RGCs diminished to 40% by 3d and to 16% by 30d without further loss. Cones diminished to 58% and 35% by 3 and 7d, respectively and further decreased between 30 and 90d. HZs showed normal values throughout the study. In conclusion, AOHT affects both the inner and outer retina, with a more pronounced degeneration of the cone than the rod pathway., (© 2020 Elsevier B.V. All rights reserved.)

Glaucoma is an age-related neurodegenerative disease that begins at the onset of aging. In this disease, there is an involvement of the immune system and therefore of the microglia. The purpose of this study is to evaluate the microglial activation using a mouse model of ocular hypertension (OHT) at the onset of aging. For this purpose, we used both naive and ocular hypertensives of 15-month-old mice (early stage of aging). In the latter, we analyzed the OHT eyes and the eyes contralateral to them to compare them with their aged controls. In the eyes of aged naive, aged OHT and aged contralateral eyes, microglial changes were observed compared to the young mice, including: (i) aged naive vs young naive: An increased soma size and vertical processes; (ii) aged OHT eyes vs young OHT eyes: A decrease in the area of the retina occupied by Iba-1 cells and in vertical processes; and (iii) aged contralateral vs young contralateral: A decrease in the soma size and arbor area and an increase in the number of microglia in the outer segment layer. Aged OHT eyes and the eyes contralateral to them showed an up-regulation of the CD68 expression in the branched microglia and a down-regulation in the MHCII and P2RY12 expression with respect to the eyes of young OHT mice. Conclusion: in the early phase of aging, morphological microglial changes along with changes in the expression of MHCII, CD68 and P2RY12, in both naive and OHT mice. These changes appear in aged OHT eyes and the eyes contralateral to them eyes., (© 2020 Elsevier B.V. All rights reserved.)

Glaucoma is a common cause of visual impairment and blindness, characterized by retinal ganglion cell (RGC) death. The mechanisms that trigger the development of glaucoma remain unknown and have gained significant relevance in the study of this neurodegenerative disease. P2X7 purinergic receptors (P2X7R) could be involved in the regulation of the synaptic transmission and neuronal death in the retina through different pathways. The aim of this study was to characterize the molecular signals underlying glaucomatous retinal injury. The time-course of functional, morphological, and molecular changes in the glaucomatous retina of the DBA/2J mice were investigated. The expression and localization of P2X7R was analysed in relation with retinal markers. Caspase-3, JNK, and p38 were evaluated in control and glaucomatous mice by immunohistochemical and western-blot analysis. Furthermore, electroretinogram recordings (ERG) were performed to assess inner retina dysfunction. Glaucomatous mice exhibited changes in P2X7R expression as long as the pathology progressed. There was P2X7R overexpression in RGCs, the primary injured neurons, which correlated with the loss of function through ERG measurements. All analyzed MAPK and caspase-3 proteins were upregulated in the DBA/2J retinas suggesting a pro-apoptotic cell death. The increase in P2X7Rs presence may contribute, together with other factors, to the changes in retinal functionality and the concomitant death of RGCs. These findings provide evidence of possible intracellular pathways responsible for apoptosis regulation during glaucomatous degeneration., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

The immune system plays an important role in glaucomatous neurodegeneration. Retinal microglial reactivation associated with ganglion cell loss could reportedly contribute to the glaucoma progression. Recently we have described signs of microglia activation both in contralateral and ocular hypertension (OHT) eyes involving all retinal layers 15 days after OHT laser induction in mice. However, no works available have analyzed the microglial activation at earliest time points after OHT induction (24 h) in this experimental model. Thus, we seek to describe and quantify signs of microglia activation and differences depending on the retinal layer, 24 h after unilateral laser-induced OHT. Two groups of adult Swiss mice were used: age-matched control (naïve) and lasered. In the lasered animals, OHT eyes as well as contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1 and MHC-II. We quantified the number of microglial cells in the photoreceptor layer (OS), outer plexiform layer (OPL), and inner plexiform layer (IPL); the number of microglial vertical processes connecting the OPL and OS; the area of the retina occupied by Iba-1+ cells (Iba1-RA) in the nerve fiber layer-ganglion cell layer (NFL-GCL), the total arbor area of microglial cells in the OPL and IPL and; Iba-1+ cell body area in the OPL, IPL and NFL-GCL. In contralateral and OHT eyes the morphological features of Iba-1+ cell activation were: migration, enlargement of the cell body, higher degree of branching and reorientation of the processes, radial disposition of the soma and processes toward adjacent microglial plexuses, and presence of amoeboid cells acting as macrophages. These signs were more pronounced in OHT eyes. Most of Iba-1+ cells did not express MHC-II; rather, only dendritic and rounded cells expressed it. In comparison with naïve eyes, in OHT eyes and contralateral eyes no significant differences were found in the microglial cell number; but there was a significant increase in Iba1-RA. The total arbor area of microglial cells was significantly decreased in: i) OHT eyes with respect contralateral eyes and naïve-eyes in IPL; ii) OHT eyes with respect to naïve eyes in OPL. The number of microglial vertical processes connecting the OPL and OS were significantly increased in contralateral eyes compared with naïve-eyes and OHT eyes. In OPL, IPL and NFL-GCL, the cell body area of Iba-1+ cells was significantly greater in OHT eyes than in naïve and contralateral eyes, and greater in contralateral eyes than in naïve eyes. A non-proliferative microglial reactivation was detected both in contralateral eyes and in OHT eyes in an early time after unilateral laser-induced OHT (24 h). This fast microglial activation, which involves the contralateral eye, could be mediated by the immune system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We investigated the feasibility and efficacy of using a specific sphingosine 1-phosphate (S1P1) receptor agonist, CYM-5442, to slow or block retinal ganglion cell (RGC) loss in endothelin-1 (ET-1) induced RGC loss. A single intravitreal injection of ET-1 (20pmol/ul), a potent vasoactive peptide that produces retinal vessels vasoconstriction, was used to induce and characterize RGC-specific cell death. CYM-5442 (1 mgr/kg) or vehicle was administered intraperitoneally for five consecutive days after ET-1-induced RGC loss. The functional extent of RGC loss injury was evaluated with pattern visual evoked potentials (VEP) and electroretinography. RGCs and retinal nerve fiber layer (RNFL) thickness were assessed in vivo using optical coherence tomography and ex vivo using Brn3a immunohistochemistry in flat-mounted retinas. ET-1 caused significant RGC loss and function loss one week after intravitreal injection. VEP showed preserved visual function after CYM-5442 administration compared to vehicle-treated animals (11.95 ± 0.86 μV vs 3.47 ± 1.20 μV, n = 12) (p < 0.05). RNFL was significantly thicker in the CYM treated-animals compared to the vehicle (93.62 ± 3.22 μm vs 77.72 ± 0.35 μm, n = 12) (p < 0.05). Furthermore, Brn3a immunohistochemistry validated this observation, showing significantly higher RGCs numbers in CYM treated rats than in the vehicle group (76,540 ± 303 vs 52,426 ± 1,932 cells/retina, n = 9) (p = 0.05). CYM-5442 administration was associated with significant retinal cleaved caspase-3 deactivation, indicating reduced apoptotic levels. The results of the present study further demonstrate the important role of S1P1 receptor agonists to lessen intravitreal ET-1 induced RGC loss., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

To study the effects of ocular hypertension (OHT) on the visual system of C57BL/6 pigmented mice, the limbal and episcleral veins of the left eye were laser photocoagulated (LP). LP increased the intraocular pressure during the first five days (d), reaching basal values at 7d. To investigate the effect of OHT on the retinal ganglion cell (RGC) retrograde axonal transport, hydroxistilbamidine methanesulfonate (OHSt) was applied to both superior colliculi (SCi) and the retinas were dissected 2 or 4 weeks after LP. To determine RGC survival, these same retinas were immunoreacted against Brn3a (general RGC population) and melanopsin (intrinsically photosensitive RGCs, m+RGCs). To study whether OHT affected non-RGC neurons in the ganglion cell layer (GCL), RGCs were immunodetected with Brn3a and all GCL nuclei counterstained with DAPI in a group of animals examined 4 weeks post-LP. Innervation of the SCi was examined at 10 days, 8 or 14 weeks after LP with the orthogradely transported cholera toxin subunit-B. OHT resulted in diffuse and sectorial loss of OHSt+RGCs (50% at 2 weeks and 62% at 4 weeks) and in a comparable loss of Brn3a+RGCs at the same time intervals. m+RGCs decreased to 59% at 2 weeks and to 46% at 4 weeks, such loss was diffuse, did not parallel the sectorial loss of the general RGC population and was more severe in the superior-temporal retina. In the GCL, cell loss is selective for RGCs and does not affect other non-RGC neurons. The retinotectal innervation appeared significantly reduced at 10 days (55.7%) and did not progress further up to 14 weeks (46.6%). Thus, LP-induced OHT results in retrograde degeneration of RGCs and m+RGCs, as well as in the loss of CTB-labelled retinotectal terminals.

To investigate the long-term effects of laser-photocoagulation (LP)-induced ocular hypertension (OHT) in the innermost and outermost (outer-nuclear and outer segment)-retinal layers (ORL). OHT was induced in the left eye of adult rats. To investigate the ganglion cell layer (GCL) wholemounts were examined at 1, 3 or 6 months using Brn3a-immunodetection to identify retinal ganglion cells (RGCs) and DAPI-staining to detect all nuclei in this layer. To study the effects of LP on the ORL up to 6 months, retinas were: i) fresh extracted to quantify the levels of rod-, S- and L-opsin; ii) cut in cross-sections for morphometric analysis, or; iii) prepared as wholemounts to quantify and study retinal distributions of entire populations of RGCs (retrogradely labeled with fluorogold, FG), S- and L-cones (immunolabeled). OHT resulted in wedge-like sectors with their apex on the optic disc devoid of Brn3a(+)RGCs but with large numbers of DAPI(+)nuclei. The levels of all opsins diminished by 2 weeks and further decreased to 20% of basal-levels by 3 months. Cross-sections revealed focal areas of ORL degeneration. RGC survival at 15 days represented approximately 28% and did not change with time, whereas the S- and L-cone populations diminished to 65% and 80%, or to 20 and 35% at 1 or 6 months, respectively. In conclusion, LP induces in the GCL selective RGCs loss that does not progress after 1 month, and S- and L-cone loss that progresses for up to 6 months. Thus, OHT results in severe damage to both the innermost and the ORL., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In rats and mice, limbar tissues of the left eye were laser-photocoagulated (LP) and ocular hypertension (OHT) effects were investigated 1 week to 6 months later. To investigate the innermost layers, retinas were examined in wholemounts using tracing from the superior colliculi to identify retinal ganglion cells (RGCs) with intact retrograde axonal transport, melanopsin immunodetection to identify intrinsically photosensitive RGCs (m(+)RGC), Brn3a immunodetection to identify most RGCs but not m(+)RGCs, RECA1 immunodetection to examine the inner retinal vessels, and DAPI staining to detect all nuclei in the GC layer. The outer retinal layers (ORLs) were examined in cross sections analyzed morphometrically or in wholemounts to study S- and L-cones. Innervation of the superior colliculi was examined 10 days to 14 weeks after LP with orthogradely transported cholera toxin subunit B. By 2 weeks, OHT resulted in pie-shaped sectors devoid of FG(+)RGCs or Brn3a(+)RGCs but with large numbers of DAPI(+)nuclei. Brn3a(+)RGCs were significantly greater than FG(+)RGCs, indicating the survival of large numbers of RGCs with their axonal transport impaired. The inner retinal vasculature showed no abnormalities that could account for the sectorial loss of RGCs. m(+)RGCs decreased to approximately 50-51% in a diffuse loss across the retina. Cross sections showed focal areas of degeneration in the ORLs. RGC loss at 1m diminished to 20-25% and did not progress further with time, whereas the S- and L-cone populations diminished progressively up to 6m. The retinotectal projection was reduced by 10 days and did not progress further. LP-induced OHT results in retrograde degeneration of RGCs and m(+)RGCs, severe damage to the ORL, and loss of retinotectal terminals., (© 2015 Elsevier B.V. All rights reserved.)

Background: Glaucomatous optic neuropathy, a leading cause of blindness, can progress despite control of intraocular pressure - currently the main risk factor and target for treatment. Glaucoma progression shares mechanisms with neurodegenerative disease, including microglia activation. In the present model of ocular hypertension (OHT), we have recently described morphological signs of retinal microglia activation and MHC-II upregulation in both the untreated contralateral eyes and OHT eyes. By using immunostaining, we sought to analyze and quantify additional signs of microglia activation and differences depending on the retinal layer., Methods: Two groups of adult Swiss mice were used: age-matched control (naïve, n = 12), and lasered (n = 12). In the lasered animals, both OHT eyes and contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1, MHC-II, CD68, CD86, and Ym1. The Iba-1+ cell number in the plexiform layers (PL) and the photoreceptor outer segment (OS), Iba-1+ arbor area in the PL, and area of the retina occupied by Iba-1+ cells in the nerve fiber layer-ganglion cell layer (NFL-GCL) were quantified., Results: The main findings in contralateral eyes and OHT eyes were: i) ameboid microglia in the NFL-GCL and OS; ii) the retraction of processes in all retinal layers; iii) a higher level of branching in PL and in the OS; iv) soma displacement to the nearest cell layers in the PL and OS; v) the reorientation of processes in the OS; vi) MHC-II upregulation in all retinal layers; vii) increased CD68 immunostaining; and viii) CD86 immunolabeling in ameboid cells. In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL. In addition, rounded Iba-1+ CD86+ cells in the NFL-GCL, OS and Ym1+ cells, and rod-like microglia in the NFL-GCL were restricted to OHT eyes., Conclusions: Several quantitative and qualitative signs of microglia activation are detected both in the contralateral and OHT eyes. Such activation extended beyond the GCL, involving all retinal layers. Differences between the two eyes could help to elucidate glaucoma pathophysiology.

The DBA/2J mouse is a model of ocular hypertension and retinal ganglion cell (RGC) degeneration, the main features of which are iris pigment dispersion (IPD) and iris stromal atrophy (ISA). These animals also experience glaucomatous changes, including an increase in intraocular pressure (IOP) beginning at about 9-12 months of age and sectorial RGC death in the retina. The aim of this study was to determine the onset of functional changes exhibited by DBA/2J mice in the inner retina. This was performed by means of electroretinographic recordings (scotopic threshold response, STR) and their correlation with morphological changes (loss of RGCs). To this end, we recorded the scotopic threshold response in control C57BL/6J and in DBA/2J mice at different ages. The RGCs, in both DBA/2J and C57BL/6J animals, were identified at 15 months of age by retrograde tracing with an analogue of fluorogold, hydroxystilbamidine methanesulfonate (OHSt), applied on the superior colliculi. Whole mount retinas were processed to quantify the population of RGCs identified by fluorogold tracing and Brn3a immunodetection, and were counted using image analysis software; an isodensity contour plot was generated for each retina. DBA/2J mice showed a significant reduction in the positive STR (pSTR) amplitudes at 12 months of age, as compared to control C57BL/6J mice of the same age. The pSTR mean amplitude decreased to approximately 27.82% of the values recorded in control mice (p = 0.0058). STR responses decreased in both strains as a result of the natural process of aging, but the decrease was more pronounced in DBA/2J mice. Furthermore, quantification of the total number of RGCs identified by OHSt and Brn3a expression showed a reduced population of RGCs in DBA/2J mice as compared to control mice. Regression analysis revealed significant correlations between the decrease in pSTR and a non-homogeneous reduction in the number of RGCs throughout the retina. Our results indicate the existence of a correlation between retinal function impairment and RGC loss. This functional and morphological analysis allows a reliable assessment of the progression of the disease., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In the mouse model of unilateral laser-induced ocular hypertension (OHT) the microglia in both the treated and the normotensive untreated contralateral eye have morphological signs of activation and up-regulation of MHC-II expression in comparison with naïve. In the brain, rod-like microglia align to less-injured neurons in an effort to limit damage. We investigate whether: i) microglial activation is secondary to laser injury or to a higher IOP and; ii) the presence of rod-like microglia is related to OHT. Three groups of mice were used: age-matched control (naïve, n=15); and two lasered: limbal (OHT, n=15); and non-draining portion of the sclera (scleral, n=3). In the lasered animals, treated eyes as well as contralateral eyes were analysed. Retinal whole-mounts were immunostained with antibodies against, Iba-1, NF-200, MHC-II, CD86, CD68 and Ym1. In the scleral group (normal ocular pressure) no microglial signs of activation were found. Similarly to naïve eyes, OHT-eyes and their contralateral eyes had ramified microglia in the nerve-fibre layer related to the blood vessel. However, only eyes with OHT had rod-like microglia that aligned end-to-end, coupling to form trains of multiple cells running parallel to axons in the retinal surface. Rod-like microglia were CD68+ and were related to retinal ganglion cells (RGCs) showing signs of degeneration (NF-200+RGCs). Although MHC-II expression was up-regulated in the microglia of the NFL both in OHT-eyes and their contralateral eyes, no expression of CD86 and Ym1 was detected in ramified or in rod-like microglia. After 15 days of unilateral lasering of the limbal and the non-draining portion of the sclera, activated microglia was restricted to OHT-eyes and their contralateral eyes. However, rod-like microglia were restricted to eyes with OHT and degenerated NF-200+RGCs and were absent from their contralateral eyes. Thus, rod-like microglia seem be related to the neurodegeneration associated with HTO.

Non-viral gene therapy represents a promising approach for the treatment of retinal diseases. However, the lack of an efficient carrier hampers the implementation of this therapy. In this study, we evaluated low molecular weight ultrapure oligochitosans for the delivery of the pCMS-EGFP plasmid into the rat retina cells after subretinal and intravitreal administrations. Polyplexes were technologically characterized. Resulting polyplexes based on ultrapure oligochitosans were slightly spherical, protected the plasmid against enzymatic digestion, and their charge and size values ranged from 8 to 14 millivolts and from 150 to 69 nm respectively depending on the N/P ratio. In HEK-293 cultured cells, transfection efficiency significantly increased from 12% to 30% when pH decreased from 7.4 to 7.1 (data normalized to Lipofectamine™ 2000). However, no significant transfection was detected in ARPE-19 cultured cells. Subretinal administrations transfected mainly the pigmented cells of the retinal pigment epithelium and the light sensitive photoreceptor cells, whereas intravitreal injections transfected cells in the ganglion cell layer, blood vessels in the inner layers of the retina and photoreceptors. These results support the potential use of oligochitosans for delivering genetic material into retinal cells in vivo., (Copyright © 2012. Published by Elsevier B.V.)

Purpose: To investigate the anatomic and functional changes triggered by light exposure in the albino mouse retina and compare them with those observed in the albino rat., Methods: BALB/c albino mice were exposed to 3,000 lx of white light during 24 h and their retinas analyzed from 1 to 180 days after light exposure (ALE). Left pupil mydriasis was induced with topical atropine. Retinal function was analyzed by electroretinographic (ERG) recording. To assess retinal degeneration, hematoxylin and eosin staining, the TdT-mediated dUTP nick-end labeling (TUNEL) technique, and quantitative immunohistofluorescence for synaptophysin and protein kinase Cα (PKCα) were used in cross sections. Intravenous injection of horseradish peroxidase and Fluoro-Gold™ tracing were used in whole-mounted retinas to study the retinal vasculature and the retinal ganglion cell (RGC) population, respectively., Results: Light exposure caused apoptotic photoreceptor death in the central retina. This death was more severe in the dorsal than in the ventral retina, sparing the periphery. Neither retinal vascular leakage nor retinal ganglion cell death was observed ALE. The electroretinographic a-wave was permanently impaired, while the b-wave decreased but recovered gradually by 180 days ALE. The scotopic threshold responses, associated with the inner retinal function, diminished at first but recovered completely by 14 days ALE. This functional recovery was concomitant with the upregulation of protein kinase Cα and synaptophysin. Similar results were obtained in both eyes, irrespective of mydriasis., Conclusions: In albino mice, light exposure induces substantial retinal damage, but the surviving photoreceptors, together with compensatory morphological/molecular changes, allow an important restoration of the retinal function.

Glaucoma, the second most common cause of blindness, is characterized by a progressive loss of retinal ganglion cells and their axons, with a concomitant loss of the visual field. Although the exact pathogenesis of glaucoma is not completely understood, a critical risk factor is the elevation, above normal values, of the intraocular pressure. Consequently, deciphering the anatomical and functional changes occurring in the rodent retina as a result of ocular hypertension has potential value, as it may help elucidate the pathology of retinal ganglion cell degeneration induced by glaucoma in humans. This paper predominantly reviews the cumulative information from our laboratory's previous, recent and ongoing studies, and discusses the deleterious anatomical and functional effects of ocular hypertension on retinal ganglion cells (RGCs) in adult rodents. In adult rats and mice, perilimbar and episcleral vein photocauterization induces ocular hypertension, which in turn results in devastating damage of the RGC population. In wide triangular sectors, preferentially located in the dorsal retina, RGCs lose their retrograde axonal transport, first by a functional impairment and after by mechanical causes. This axonal damage affects up to 80% of the RGC population, and eventually causes their death, with somal and intra-retinal axonal degeneration that resembles that observed after optic nerve crush. Importantly, while ocular hypertension affects the RGC population, it spares non-RGC neurons located in the ganglion cell layer of the retina. In addition, functional and morphological studies show permanent alterations of the inner and outer retinal layers, indicating that further to a crush-like injury of axon bundles in the optic nerve head there may by additional insults to the retina, perhaps of ischemic nature., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: To analyze the effects of different levels of intraocular pressure (IOP) in the macroglia in ocular hypertension (OHT) and contralateral eyes at 3 weeks after laser photocoagulation and compare these with effects in age-matched control rats., Methods: Adult Sprague-Dawley rats were divided into an age-matched control (naive) group and an OHT group. Retinas were processed as whole mounts and immunostained with GFAP for analysis of the retinal macroglia., Results: The area of the retina occupied by astrocytes (AROA) was quantified. GFAP immunostaining showed common features in ipsilateral and contralateral eyes. First, although the astrocyte network maintained a star-shaped morphology, these cells had fewer secondary processes and thinner cell bodies and primary processes than did naive cells. Second, Müller cells appeared as punctate GFAP+ structures among astrocytes. Third, there was a significant reduction of the AROA in ipsilateral and contralateral eyes compared with naive eyes. Ipsilateral eyes had significantly less AROA than did contralateral eyes. The decrease was greater for OHT eyes with higher IOP levels., Conclusions: OHT induces changes in the macroglia of contralateral eyes; thus, these fellow eyes should not be used as control. In eyes with OHT, there is a close relationship between IOP values and decreased AROA.

Optic nerve transection (ONT) triggers retinal ganglion cell (RGC) death. By using this paradigm, we have analyzed for the first time in adult albino and pigmented mice, the effects of ONT in the scotopic threshold response (STR) components (negative and positive) of the full-field electroretinogram. Two weeks after ONT, when in pigmented mice approximately 18% of the RGC population survive, the STR-implicit time decreased and the p and nSTR waves diminished approximately to 40% or 55%, in albino or pigmented, respectively, with respect to the values recorded from the non-operated contralateral eyes. These changes were maintained up to 12 weeks post-ONT, demonstrating that the ERG-STR is a useful parameter to monitor RGC functionality in adult mice., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In adult albino mice the effects of increased intraocular pressure on the outer retina and its circuitry was investigated at intervals ranging 3-14 weeks. Ocular hypertension (OHT) was induced by cauterizing the vessels draining the anterior part of the mice eye, as recently reported (Salinas-Navarro et al., 2009a). Electroretinographic (ERG) responses were recorded simultaneously from both eyes and compared each other prior to and at different survival intervals of 2, 8 or 12 weeks after lasering. Animals were processed at 3, 9 or 14 weeks after lasering, and radial sections were obtained in the cryostat and further processed for immunocytochemistry using antibodies against recoverin, gamma-transducin, Protein Kinase C-alpha (PKC-alpha), calbindin or synaptophysin. The synaptic ribbons were identified using an antibody against the protein bassoon, which labels photoreceptor ribbons and nuclei were identified using TO-PRO. Laser photocoagulation of the perilimbar and episcleral veins of the left eye resulted in an increase in mean intraocular pressure to approximately over twice its baseline by 24 h that was maintained for approximately five days reaching basal levels by 1 week. ERG recordings from the different groups of mice showed their a-, b-wave and scotopic threshold response (STR) amplitudes, when compared to their contralateral fellow eye, reduced to 62%, 52% and 23% at 12 weeks after lasering. Three weeks after lasering, immunostaining with recoverin and transducin antibodies could not document any changes in the outer nuclear layer (ONL) but both ON-rod bipolar and horizontal cells had lost their dendritic processes in the outer plexiform layer (OPL). Sprouting of horizontal and bipolar cell processes were observed into the ONL. Fourteen weeks after lasering, protein kinase-C antibodies showed morphologic changes of ON-rod bipolar cells and calbindin staining showed abnormal horizontal cells and a loss of their relationship with their presynaptic input. Moreover, at this time, quantitative studies indicate significant diminutions in the number of photoreceptor synaptic ribbons/100 microm, and in the thickness of the outer nuclear and plexiform layer, when compared to their fellow eyes. Increased intraocular pressure in Swiss mice results in permanent alterations of their full field ERG responses and in changes of the inner and outer retinal circuitries., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Ocular hypertension (OHT) is the main risk factor of glaucoma, a neuropathy leading to blindness. Here we have investigated the effects of laser photocoagulation (LP)-induced OHT, on the survival and retrograde axonal transport (RAT) of adult rat retinal ganglion cells (RGC) from 1 to 12 wks. Active RAT was examined with fluorogold (FG) applied to both superior colliculi (SCi) 1 wk before processing and passive axonal diffusion with dextran tetramethylrhodamine (DTMR) applied to the optic nerve (ON) 2 d prior to sacrifice. Surviving RGCs were identified with FG applied 1 wk pre-LP or by Brn3a immunodetection. The ON and retinal nerve fiber layer were examined by RT97-neurofibrillar staining. RGCs were counted automatically and color-coded density maps were generated. OHT retinas showed absence of FG+ or DTMR+RGCs in focal, pie-shaped and diffuse regions of the retina which, by two weeks, amounted to, approximately, an 80% of RGC loss without further increase. At this time, there was a discrepancy between the total number of surviving FG-prelabelled RGCs and of DMTR+RGCs, suggesting that a large proportion of RGCs had their RAT impaired. This was further confirmed identifying surviving RGCs by their Brn3a expression. From 3 weeks onwards, there was a close correspondence of DTMR+RGCs and FG+RGCs in the same retinal regions, suggesting axonal constriction at the ON head. Neurofibrillar staining revealed, in ONs, focal degeneration of axonal bundles and, in the retinal areas lacking backlabeled RGCs, aberrant staining of RT97 characteristic of axotomy. LP-induced OHT results in a crush-like injury to ON axons leading to the anterograde and protracted retrograde degeneration of the intraocular axons and RGCs.

Purpose: To investigate the effects of laser photocoagulation (LP)-induced ocular hypertension (OHT) on the survival and retrograde axonal transport of retinal ganglion cells (RGC), as well as on the function of retinal layers., Methods: Adult albino Swiss mice (35-45 g) received laser photocoagulation of limbal and episcleral veins in the left eye. Mice were sacrificed at 8, 17, 35, and 63 days. Intraocular pressure (IOP) in both eyes was measured with a Tono-Lab before LP and at various days after LP. Flash electroretinogram (ERG) scotopic threshold response (STR) and a- and b-wave amplitudes were recorded before LP and at various times after LP. RGCs were labeled with 10% hydroxystilbamidine methanesulfonate (OHSt) applied to both superior colliculi before sacrifice and in some mice, with dextran tetramethylrhodamine (DTMR) applied to the ocular stump of the intraorbitally transected optic nerve. Retinas were immunostained for RT97 or Brn3a. Retinas were prepared as whole-mounts and photographed under a fluorescence microscope. Labeled RGCs were counted using image analysis software, and an isodensity contour plot was generated for each retina., Results: IOP increased to twice its basal values by 24 h and was maintained until day 5, after which IOP gradually declined to reach basal values by 1 wk. Similar IOP increases were observed in all groups. The mean total number of OHSt(+) RGCs was 13,428+/-6,295 (n=12), 10,456+/-14,301 (n=13), 12,622+/-14,174 (n=21), and 10,451+/-13,949 (n=13) for groups I, II, III, and IV, respectively; these values represented 28%, 23%, 26%, and 22% of the values found in their contralateral fellow retinas. The mean total population of Brn3a(+) RGCs was 24,343+/-5,739 (n=12) and 10,219+/-8,887 (n=9), respectively, for groups I and III; these values represented 49% and 20%, respectively, of the values found in their fellow eyes. OHT retinas showed an absence of OHSt(+) and DTMR(+) RGCs in both focal wedge-shaped and diffuse regions of the retina. By 1 wk, there was a discrepancy between the total number of surviving OHSt(+) RGCs and Brn3a(+) RGCs, suggesting that a large proportion of RGCs had impaired retrograde axonal transport. In the retinal areas lacking backlabeled RGCs, neurofibrillar staining revealed aberrant expression of RT97 within axons and RGC bodies characteristic of axotomy. Elevated IOP induced significant reductions in the registered ERG waves, including positive STR, a- and b-waves, that were observed by 24 h and remained throughout the period of study for the three groups analyzed., Conclusions: LP of the perilimbal and episcleral veins resulted in OHT leading to a lack of retrograde axonal transport in approximately 75% of the original RGC population. This lack did not progress further between 8 and 63 days, and it was both focal (in sectors with the apex located in the optic disc) and diffuse within the retina. In addition, severe amplitude diminutions of the STR and a- and b-waves of the ERG appeared as early as 24 h after lasering and did not recover throughout the period of study, indicating that increased IOP results in severe damage to the innermost, inner nuclear, and outer nuclear layers of the retina.

Purpose: To investigate the different components of full-field flash electroretinogram (ERG) responses in adult albino and pigmented rats at various time intervals following optic nerve transection (ONT)., Methods: In adult Sprague-Dawley (SD, albino) and Piebald-Viral-Glaxo (PVG, pigmented) rats, the left optic nerve was transected intraorbitally to induce retinal ganglion cell (RGC) death. ERG responses were recorded simultaneously from both eyes beforehand and at 1, 2, 4, and 12 week intervals after ONT. The ERG a- and b-waves and the scotopic threshold responses (STR) were analyzed in scotopic conditions. White light stimuli of intensities ranging from 10(-6) to 10(-4) cd.s.m(-2) were used to record the positive and negative scotopic threshold responses (pSTR and nSTR), while stimulus light intensities ranging from 10(-4) to 10(2) cd.s.m(-2) were used to analyze the a- and b-wave amplitudes of standard ERG recordings., Results: In the albino rats, one week after intraorbital ONT, the STR mean amplitude decreased significantly, to approximately 60% of the values registered for the contralateral eye (p<0.05), which had not been operated on; standard ERG a- and b-waves showed a small reduction in amplitude-to approximately 85%. By two weeks after ONT, the STR mean amplitude was approximately 40% that of the contralateral eye, while the a- and b-wave amplitudes had further decreased to approximately 75%. Four weeks after ONT, the STR had fallen to 60% of that of the contralateral eyes, whereas the a- and b-waves reached values of approximately 90%. Twelve weeks after ONT, the STR remained significantly reduced to approximately 45%, whereas the a- and b-waves reached values of approximately 90%. In the pigmented rats, one week after intraorbital ONT, the mean amplitude had decreased significantly, to approximately 60% for the pSTR and to 80% for the nSTR of the values registered for the intact contralateral eye (p<0.05); while the standard ERG a- and b-waves showed a small reduction in amplitude to approximately 90%. Two weeks after ONT, the STR mean amplitude was approximately 55%, while the a- and b-wave amplitudes had further decreased to approximately 65%. Four weeks after ONT, the STR also was significantly reduced, to only 40%, whereas the a- and b-waves reached values of approximately 60%. Twelve weeks after ONT, the pSTR and nSTR remained significantly reduced to approximately 40% and 70%, respectively; whereas the a- and b-waves reached values of approximately 80%., Conclusions: Optic nerve injury results in transient reductions of the major ERG components, the a- and b-waves, as well as permanent reductions of the early components of the ERG, the negative and positive scotopic threshold responses. Because ONT induces massive RGC loss, it is likely that permanent reduction in the STR represents a lack of the RGC population, thus highlighting the importance of the STR recordings as an electrophysiological tool for the assessment of RGC function.

Purpose: To investigate the long-term effects of transient ligature of the ophthalmic vessels (LOV) on the inner and outer retina as well as on retinotectal projection, and whether brimonidine (BMD) has protective effects., Methods: In adult rats, the left eye was subjected to 90 minutes of LOV. One hour before ischemia, 2 drops of saline alone (vehicle group) or saline containing 0.5% brimonidine (BMD group) were instilled in the left eye. The effects of LOV on the inner and outer retina were assessed with ERG recordings of a- and b-wave amplitudes at 1, 8, and 12 weeks after LOV and with analysis of layer thickness in paraffin sections. The retinotectal projection was orthogradely labeled with cholera toxin subunit B (CTB) injected in the left eye and measured in serial coronal sections of the superior colliculus., Results: There were significant reductions in the mean b-wave amplitudes of the ischemic eyes at 8 and 12 weeks after LOV in the vehicle-treated group of animals, but not in the BMD-treated group. The thickness of the inner nuclear and inner plexiform layers of the vehicle-treated group of retinas had decreased to approximately 71% of the thicknesses in the BMD-treated groups. Three months after LOV, the mean volume of the retinotectal projection in the vehicle- or BMD-treated group of animals had decreased to approximately 54% or 83%, respectively, of the mean values found in the control group of animals., Conclusions: LOV induces degeneration of the inner retinal layers and the retinotectal projection 3 months after the insult. BMD administration significantly protected against LOV-induced retinal damage and degeneration of retinal projection.

In adult rats, we have induced retinal ischemia and investigated anterogradely labeled surviving retinal ganglion cell (RGC) afferents to the contralateral superior colliculus (SC). The animals received topically in their left eyes two 5-microl drops of saline or saline-containing 0.5% brimonidine (BMD), 1 h before 90 min of retinal ischemia induced by ligature of the left ophthalmic vessels. Two months after ischemia, the anterogradely transported neuronal tracer cholera toxin B subunit (CTB) was injected in the ischemic eyes and animals were processed 4 days later. As controls and for comparison, the retinotectal innervation of unlesioned age-matched control rats was also examined with CTB. In control and experimental animals, serial coronal sections of the mesencephalon and brainstem were immunoreacted for CTB and the area and thickness of the two most superficial layers of the SC containing densely CTB-labeled profiles were estimated with an image analysis system. Ninety minutes of ischemia resulted 2 months later in reduced density of CTB-labeled profiles in the contralateral SC of the vehicle-treated rats, representing less than one half the area occupied by CTB-labeled profiles in control rats. This resulted in shrinkage of these layers and in the presence of areas virtually devoid of CTB immunoreactivity, suggesting orthograde degeneration of retinal terminals and/or decrease of anterograde axonal transport. Topical pretreatment with BMD resulted 2 months later in CTB immunoreactivity that occupied the superficial layers of the contralateral SC in an area of approximately 86% of that observed in the unlesioned control group of animals, indicating that BMD protects against ischemia-induced degeneration of the retinotectal projection, and preserves anterograde axonal transport.