Evaluation of the neuroprotective efficacy of the gramine derivative ITH12657 against NMDA-induced excitotoxicity in the rat retina.

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.

Alpha retinal ganglion cells in pigmented mice retina: number and distribution.

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.

7,8-Dihydroxiflavone Maintains Retinal Functionality and Protects Various Types of RGCs in Adult Rats with Optic Nerve Transection.

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.

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-ß 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-1ß 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.

Arrangement of the photoreceptor mosaic in a diabetic rat model imaged with multiphoton microscopy.

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.

Functional and morphological alterations in a glaucoma model of acute ocular hypertension.

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.

Microglial changes in the early aging stage in a healthy retina and an experimental glaucoma model.

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.

Time course of bilateral microglial activation in a mouse model of laser-induced glaucoma.

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.

A Chronic Ocular-Hypertensive Rat Model induced by Injection of the Sclerosant Agent Polidocanol in the Aqueous Humor Outflow Pathway.

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.

Potential role of P2X7 receptor in neurodegenerative processes in a murine model of glaucoma.

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.

Bilateral early activation of retinal microglial cells in a mouse model of unilateral laser-induced experimental ocular hypertension.

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.

The S1P1 receptor-selective agonist CYM-5442 protects retinal ganglion cells in endothelin-1 induced retinal ganglion cell loss.

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.

Shared and Differential Retinal Responses against Optic Nerve Injury and Ocular Hypertension.

Glaucoma, one of the leading causes of blindness worldwide, affects primarily retinal ganglion cells (RGCs) and their axons. The pathophysiology of glaucoma is not fully understood, but it is currently believed that damage to RGC axons at the optic nerve head plays a major role. Rodent models to study glaucoma include those that mimic either ocular hypertension or optic nerve injury. Here we review the anatomical loss of the general population of RGCs (that express Brn3a; Brn3a+RGCs) and of the intrinsically photosensitive RGCs (that express melanopsin; m+RGCs) after chronic (LP-OHT) or acute (A-OHT) ocular hypertension and after complete intraorbital optic nerve transection (ONT) or crush (ONC). Our studies show that all of these insults trigger RGC death. Compared to Brn3a+RGCs, m+RGCs are more resilient to ONT, ONC, and A-OHT but not to LP-OHT. There are differences in the course of RGC loss both between these RGC types and among injuries. An important difference between the damage caused by ocular hypertension or optic nerve injury appears in the outer retina. Both axotomy and LP-OHT induce selective loss of RGCs but LP-OHT also induces a protracted loss of cone photoreceptors. This review outlines our current understanding of the anatomical changes occurring in rodent models of glaucoma and discusses the advantages of each one and their translational value.

Retinal neurodegeneration in experimental glaucoma.

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.

Effects of ocular hypertension in the visual system of pigmented mice.

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.

Laser-induced ocular hypertension in adult rats does not affect non-RGC neurons in the ganglion cell layer but results in protracted severe loss of cone-photoreceptors.

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.

Microglia in mouse retina contralateral to experimental glaucoma exhibit multiple signs of activation in all retinal layers.

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.

Assessment of inner retina dysfunction and progressive ganglion cell loss in a mouse model of glaucoma.

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.

Rod-like microglia are restricted to eyes with laser-induced ocular hypertension but absent from the microglial changes in the contralateral untreated eye.

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.

Oligochitosan polyplexes as carriers for retinal gene delivery.

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.