Systemic treatment with 7,8-Dihydroxiflavone activates TtkB and affords protection of two different retinal ganglion cell populations against axotomy in adult rats.

PURPOSE: To analyze responses of different RGC populations to left intraorbital optic nerve transection (IONT) and intraperitoneal (i.p.) treatment with 7,8-Dihydroxyflavone (DHF), a potent selective TrkB agonist. METHODS: Adult albino Sprague-Dawley rats received, following IONT, daily i.p. injections of vehicle (1%DMSO in 0.9%NaCl) or DHF. Group-1 (n = 58) assessed at 7days (d) the optimal DHF amount (1-25 mg/kg). Group-2, using freshly dissected naïve or treated retinas (n = 28), investigated if DHF treatment was associated with TrkB activation using Western-blotting at 1, 3 or 7d. Group-3 (n = 98) explored persistence of protection and was analyzed at survival intervals from 7 to 60d after IONT. Groups 2-3 received daily i.p. vehicle or DHF (5 mg/kg). Retinal wholemounts were immunolabelled for Brn3a and melanopsin to identify Brn3a+RGCs and m+RGCs, respectively. RESULTS: Optimal neuroprotection was achieved with 5 mg/kg DHF and resulted in TrkB phosphorylation. The percentage of surviving Brn3a+RGCs in vehicle treated rats was 60, 28, 18, 13, 12 or 8% of the original value at 7, 10, 14, 21, 30 or 60d, respectively, while in DHF treated retinas was 94, 70, 64, 17, 10 or 9% at the same time intervals. The percentages of m+RGCs diminished by 7d-13%, and recovered by 14d-38% in vehicle-treated and to 48% in DHF-treated retinas, without further variations. CONCLUSIONS: DHF neuroprotects Brn3a + RGCs and m + RGCs; its protective effects for Brn3a+RGCs are maximal at 7 days but still significant at 21d, whereas for m+RGCs neuroprotection was significant at 14d and permanent.

Bone Marrow-Derived Mononuclear Cell Transplants Decrease Retinal Gliosis in Two Animal Models of Inherited Photoreceptor Degeneration.

Inherited photoreceptor degenerations are not treatable diseases and a frequent cause of blindness in working ages. In this study we investigate the safety, integration and possible rescue effects of intravitreal and subretinal transplantation of adult human bone-marrow-derived mononuclear stem cells (hBM-MSCs) in two animal models of inherited photoreceptor degeneration, the P23H-1 and the Royal College of Surgeons (RCS) rat. Immunosuppression was started one day before the injection and continued through the study. The hBM-MSCs were injected in the left eyes and the animals were processed 7, 15, 30 or 60 days later. The retinas were cross-sectioned, and L- and S- cones, microglia, astrocytes and Müller cells were immunodetected. Transplantations had no local adverse effects and the CD45+ cells remained for up to 15 days forming clusters in the vitreous and/or a 2-3-cells-thick layer in the subretinal space after intravitreal or subretinal injections, respectively. We did not observe increased photoreceptor survival nor decreased microglial cell numbers in the injected left eyes. However, the injected eyes showed decreased GFAP immunoreactivity. We conclude that intravitreal or subretinal injection of hBM-MSCs in dystrophic P23H-1 and RCS rats causes a decrease in retinal gliosis but does not have photoreceptor neuroprotective effects, at least in the short term. However, this treatment may have a potential therapeutic effect that merits further investigation.

Topical bromfenac transiently delays axotomy-induced retinal ganglion cell loss.

Optic nerve axotomy in rodents allows detailed studies of the effect of different treatments on the survival of central nervous system neurons, the retinal ganglion cells (RGCs). Here we have analyzed the neuroprotective effect of topical bromfenac treatment, a nonsteroidal anti-inflammatory drug (NSAID) used in clinic to ameliorate post-operative inflammation, on axotomized rat RGCs. The left optic nerve of adult rats was subjected to optic nerve crush (ONC). Half of the rats were treated with a topical instillation of saline. On the other half, immediately after the surgery, 2 drops of bromfenac (0.09% Yellox; Bausch & Lomb) were instilled, and then every 12 h until analysis. Retinas in both groups were dissected 3, 5, 7, 9 and 14 days after ONC (n = 4-8/time point/group). Toxicity of bromfenac was assessed in intact retinas treated during 14 days (n = 6). Intact untreated retinas were used as control of the RGC population. RGCs were identified by Brn3a immunodetection and automatically quantified. Our results show that bromfenac does not cause RGC loss in intact retinas. In the injured groups, the number of RGCs at 7, 9 and 14 days after the lesion was significantly higher in treated vs. untreated retinas. To our knowledge this is the first report showing that a topical treatment with a NSAIDs delays axotomy-induced RGC loss and indicates that treatment with NSAIDs could be used as conjunctive therapy in diseases that proceed with optic nerve damage.

ß-alanine supplementation induces taurine depletion and causes alterations of the retinal nerve fiber layer and axonal transport by retinal ganglion cells.

To study the effect of taurine depletion induced by ß-alanine supplementation in the retinal nerve fiber layer (RNFL), and retinal ganglion cell (RGC) survival and axonal transport. Albino Sprague-Dawley rats were divided into two groups: one group received ß-alanine supplementation (3%) in the drinking water during 2 months to induce taurine depletion, and the other group received regular water. After one month, half of the rats from each group were exposed to light. Retinas were analyzed in-vivo using Spectral-Domain Optical Coherence Tomography (SD-OCT). Prior to processing, RGCs were retrogradely traced with fluorogold (FG) applied to both superior colliculi, to assess the state of their retrograde axonal transport. Retinas were dissected as wholemounts, surviving RGCs were immunoidentified with Brn3a, and the RNFL with phosphorylated high-molecular-weight subunit of the neurofilament triplet (pNFH) antibodies. ß-alanine supplementation decreases significantly taurine plasma levels and causes a significant reduction of the RNFL thickness that is increased after light exposure. An abnormal pNFH immunoreactivity in some RGC bodies, their proximal dendrites and axons, and a further diminution of the mean number of FG-traced RGCs compared with Brn3a+RGCs, indicate that their retrograde axonal transport is affected. In conclusion, taurine depletion causes RGC loss and axonal transport impairment. Finally, our results suggest that care should be taken when ingesting ß-alanine supplements due to the limited understanding of their potential adverse effects.

Retinal Ganglion Cell Death as a Late Remodeling Effect of Photoreceptor Degeneration.

Inherited or acquired photoreceptor degenerations, one of the leading causes of irreversible blindness in the world, are a group of retinal disorders that initially affect rods and cones, situated in the outer retina. For many years it was assumed that these diseases did not spread to the inner retina. However, it is now known that photoreceptor loss leads to an unavoidable chain of events that cause neurovascular changes in the retina including migration of retinal pigment epithelium cells, formation of "subretinal vascular complexes", vessel displacement, retinal ganglion cell (RGC) axonal strangulation by retinal vessels, axonal transport alteration and, ultimately, RGC death. These events are common to all photoreceptor degenerations regardless of the initial trigger and thus threaten the outcome of photoreceptor substitution as a therapeutic approach, because with a degenerating inner retina, the photoreceptor signal will not reach the brain. In conclusion, therapies should be applied early in the course of photoreceptor degeneration, before the remodeling process reaches the inner retina.

Melanopsin+RGCs Are fully Resistant to NMDA-Induced Excitotoxicity.

We studied short- and long-term effects of intravitreal injection of N-methyl-d-aspartate (NMDA) on melanopsin-containing (m+) and non-melanopsin-containing (Brn3a+) retinal ganglion cells (RGCs). In adult SD-rats, the left eye received a single intravitreal injection of 5µL of 100nM NMDA. At 3 and 15 months, retinal thickness was measured in vivo using Spectral Domain-Optical Coherence Tomography (SD-OCT). Ex vivo analyses were done at 3, 7, or 14 days or 15 months after damage. Whole-mounted retinas were immunolabelled for brain-specific homeobox/POU domain protein 3A (Brn3a) and melanopsin (m), the total number of Brn3a+RGCs and m+RGCs were quantified, and their topography represented. In control retinas, the mean total numbers of Brn3a+RGCs and m+RGCs were 78,903 ± 3572 and 2358 ± 144 (mean ± SD; n = 10), respectively. In the NMDA injected retinas, Brn3a+RGCs numbers diminished to 49%, 28%, 24%, and 19%, at 3, 7, 14 days, and 15 months, respectively. There was no further loss between 7 days and 15 months. The number of immunoidentified m+RGCs decreased significantly at 3 days, recovered between 3 and 7 days, and were back to normal thereafter. OCT measurements revealed a significant thinning of the left retinas at 3 and 15 months. Intravitreal injections of NMDA induced within a week a rapid loss of 72% of Brn3a+RGCs, a transient downregulation of melanopsin expression (but not m+RGC death), and a thinning of the inner retinal layers.

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.

Light-induced retinal degeneration causes a transient downregulation of melanopsin in the rat retina.

In this work we study the effects of an acute light-induced retinal degeneration on the population of melanopsin positive retinal ganglion cells (m+RGCs) and the expression of the melanopsin protein in the retina. The m+RGCs may be more resistant than other RGCs to lesion, but the effects of an acute light exposure in this population are unknown. Albino rats were exposed to white light (3000 lux) continuously for 48 h and processed 0, 3, 7 or 30 days after light exposure (ALE). Whole-mounted retinas were immunodetected with antibodies against melanopsin, Brn3a, and rhodopsin to study the populations of m+RGC, Brn3a+RGC and rods (which are the most abundant photoreceptors in the rat retina). Three days ALE there was substantial rod loss in an arciform area of the superior retina and with time this loss expanded in the form of rings all throughout the retina. Light exposure did not affect the number of Brn3a+RGCs but diminished the numbers of m+RGCs. Immediately ALE there was a significant decrease in the mean number of immunodetected m+RGCs that was more marked in the superior retina. Later, the number of m+RGCs increased progressively and reached normal values one month ALE. Western blot analysis showed that melanopsin expression down-regulates shortly ALE and recovers thereafter, in accordance with the anatomical data. This study demonstrates that there is a transient downregulation of melanopsin expression in the RGCs during the first month ALE. Further studies would be needed to clarify the long-term effect of light exposure on the m+RGC population.

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.

Dose-Related Side Effects of Intravitreal Injections of Humanized Anti-Vascular Endothelial Growth Factor in Rats: Glial Cell Reactivity and Retinal Ganglion Cell Loss.

Purpose: In a previous study, we documented that the Intravitreal injections (IVIs) of bevacizumab in rats caused a retinal inflammatory response. We now study whether the IVI of other humanized anti-VEGF: ranibizumab and aflibercept also cause an inflammatory reaction in the rat retina and if it depends on the dose administered. Finally, we study whether this reaction affects retinal ganglion cell (RGC) survival. Methods: Albino Sprague-Dawley rats received a single IVI of 5 µL of PBS or ranibizumab or aflibercept at the concentration used in clinical practice (10 µg/µL or 40 µg/µL) or at a lower concentration (0.38 µg/µL and 1.5 µg/µL) calculated to obtain within the rat eye the same concentration as in the human eye in clinical practice. Others received a single 5 µL IVI of a polyclonal goat anti-rat VEGF (0.015 µg/µL) or of vehicle (PBS). Animals were processed 7 days or 1 month later. Retinal whole mounts were immunolabeled for the detection of microglial, macroglial, RGCs, and intrinsically photosensitive RGCs (ipRGCs). Fluorescence and confocal microscopy were used to examine retinal changes, and RGCs and ipRGCs were quantified automatically or semiautomatically, respectively. Results: All the injected substances including the PBS induced detectable side effects, namely, retinal microglial cell activation and retinal astrocyte hypertrophy. However, there was a greater microglial and macroglial response when the higher concentrations of ranibizumab and aflibercept were injected than when PBS, the antibody anti-rat VEGF and the lower concentrations of ranibizumab or aflibercept were injected. The higher concentration of ranibizumab and aflibercept resulted also in significant RGC death, but did not cause appreciable ipRGC death. Conclusions: The IVI of all the substances had some retinal inflammatory effects. The IVI of humanized anti-VEGF to rats at high doses cause important side effects: severe inflammation and RGC death, but not ipRGC death.

IOP induces upregulation of GFAP and MHC-II and microglia reactivity in mice retina contralateral to experimental glaucoma.

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 200 kD (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<0.001); ii) GFAP-RA was increased in contralateral (P<.05) and decreased in OHT-eyes (P <0.001); iii) the mean intensity of GFAP-IR was higher in OHT-eyes (P<0.01), and the percentage of the retinal area occupied by GFAP+ cells with higher intensity levels was increased in contralateral (P=0.05) and in OHT-eyes (P<0.01); iv) both in contralateral and in OHT-eyes, GFAP was upregulated in Müller cells and microglia was activated; v) MHC-II was upregulated on macroglia and microglia. In microglia, it was similarly expressed in contralateral and OHT-eyes. By contrast, in macroglia, MHC-II upregulation was observed mainly in astrocytes in contralateral eyes and in Müller cells in OHT-eyes; vi) NF-200+ RGCs (degenerated cells) appeared in OHT-eyes with a trend for the GFAP-RA to decrease and for the NF-200+RGC number to increase from the center to the periphery (r= -0.45). 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.

Retinal compensatory changes after light damage in albino mice.

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.

Bone marrow-derived mononuclear stem cells in the treatment of retinal degenerations.

Retinal degenerative diseases affecting the outer retina in its many forms (inherited, acquired or induced) are characterized by photoreceptor loss, and represent currently a leading cause of irreversible vision loss in the world. At present, there are very few treatments capable of preventing, recovering or reversing photoreceptor degeneration or the secondary retinal remodeling, which follows photoreceptor loss and can also cause the death of other retinal cells. Thus, these diseases are nowadays one of the greatest challenges in the field of ophthalmological research. Bone marrow derived-mononuclear stem cell transplantation has shown promising results for the treatment of photoreceptor degenerations. These cells may have the potential to slow down photoreceptor loss, and therefore should be applied in the early stages of photoreceptor degenerations. Furthermore, because of their possible paracrine effects, they may have a wide range of clinical applications, since they can potentially impact on several retinal cell types at once and photoreceptor degenerations can involve different cells and/or begin in one cell type and then affect adjacent cells. The intraocular injection of bone marrow derived-mononuclear stem cells also enhances the outcomes of other treatments aimed to protect photoreceptors. Therefore, it is likely that future investigations may combine bone marrow derived-mononuclear stem cell therapy with other systemic or intraocular treatments to obtain greater therapeutic effects in degenerative retinal diseases.

Intravitreal and subretinal syngeneic bone marrow mononuclear stem cell transplantation improves photoreceptor survival but does not ameliorate retinal function in two rat models of retinal degeneration.

PURPOSE: To study and compare effects of syngeneic bone marrow mononuclear stem cells (BM-MNCs) transplants on inherited retinal degeneration in two animal models with different etiologies: the RCS and the P23H-1 rats. To compare the safety and efficacy of two methods of intraocular delivery: subretinal and/or intravitreal. METHODS: A suspension of BM-MNCs was injected subretinally or intravitreally in the left eyes of P23H-1 and RCS rats at post-natal day (P) 21. At different survival intervals after the injection: 7, 15, 30 or 60 days, the retinas were cross-sectioned, and photoreceptor survival and glial cell responses were investigated using immunodetection of cones (anti-cone arrestin), synaptic connections (anti-bassoon), microglia (anti-Iba-1), astrocytes and Müller cells (anti-GFAP). Electroretinographic function was also assessed longitudinally. RESULTS: Intravitreal injections (IVIs) or subretinal injections (SRIs) of BM-MNCs did not produce adverse effects. The transplanted cells survived for up to 15 days but did not penetrate the retina. Both IVIs and SRIs increased photoreceptor survival, decreased synaptic degeneration and glial fibrillary acidic protein (GFAP) expression in Müller cells but did not modify microglial cell activation and migration or the electroretinographic responses. CONCLUSIONS: Intravitreal and subretinal syngeneic BM-MNCs transplantation decreases photoreceptor degeneration and shows anti-gliotic effects on Müller cells but does not ameliorate retinal function. Moreover, syngeneic BM-MNCs transplants are more effective than the xenotransplants of these cells. BM-MNC transplantation has potential therapeutic effects that merit further investigation.

Retinal ganglion cell axonal compression by retinal vessels in light-induced retinal degeneration.

PURPOSE: To analyze the damage produced by light in mydriatic and miotic albino retinas under two different sources of light. METHODS: Albino Sprague Dawley female rats were exposed to 3,000 lx during 48 h under two different light sources: linear and circular bulbs. Before exposure, their left pupils were dilated. Before and at different times after light exposure (ALE), electroretinographic signals were recorded. One week before processing, retinal ganglion cells (RGCs) were traced by applying fluorogold on the superior colliculi. Just before processing, some animals were intravenously injected with horseradish peroxidase to analyze retinal vascular leakage. At different times ALE, animals were sacrificed and their retinas dissected as whole mounts or cross-sections. Cross-sections were used to study the retinal degeneration and to detect apoptotic nuclei by the transferase dUTP nick end labeling (TUNEL) technique. Whole mounts were used to analyze vascular leakage; investigate the nerve fiber layer, identified by immunodetection of neurofilaments; and quantify the whole population of RGCs identified by fluorogold tracing and Brn3a immunodetection. With the quantitative data, detailed isodensity maps were generated to study the spatial loss of RGCs. RESULTS: Phototoxicity causes an immediate and permanent abolishment of the electroretinographic response. Early ALE, photoreceptors degenerate by apoptosis and this death is more severe in mydriatic conditions and under circular bulbs. Photoreceptor loss starts in an arciform dorsomedial retinal area, but at 3 months ALE has spread to the whole retina and there are no differences related to either pupil dilation or light source. Three months ALE, RGC axons show distorted trajectories and abnormal expression of neurofilaments. Six months or more ALE, there is significant death of RGCs caused by axonal strangulation by displaced inner retinal vessels. Topography of the surviving RGCs shows that their loss is not uniform throughout the retina. CONCLUSIONS: Light damage to photoreceptors depends on pupil dilation and light source, but affects all retinal layers with time. These deteriorative events are also observed in light-induced and inherited retinal degenerations in pigmented animals, but occur differently. Thus, the role of ocular pigmentation and the etiology of photoreceptor degeneration on retinal remodelling deserve further investigation.

Retinal ganglion cell numbers and delayed retinal ganglion cell death in the P23H rat retina.

The P23H-1 rat strain carries a rhodopsin mutation frequently found in retinitis pigmentosa patients. We investigated the progressive degeneration of the inner retina in this strain, focussing on retinal ganglion cells (RGCs) fate. Our data show that photoreceptor death commences in the ventral retina, spreading to the whole retina as the rat ages. Quantification of the total number of RGCs identified by Fluorogold tracing and Brn3a expression, disclosed that the population of RGCs in young P23H rats is significantly smaller than in its homologous SD strain. In the mutant strain, there is also RGC loss with age: RGCs show their first symptoms of degeneration at P180, as revealed by an abnormal expression of cytoskeletal proteins which, at P365, translates into a significant loss of RGCs, that may ultimately be caused by displaced inner retinal vessels that drag and strangulate their axons. RGC axonal compression begins also in the ventral retina and spreads from there causing RGC loss through the whole retinal surface. These decaying processes are common to several models of photoreceptor loss, but show some differences between inherited and light-induced photoreceptor degeneration and should therefore be studied to a better understanding of photoreceptor degeneration and when developing therapies for these diseases.

Corneal endothelial cell loss after trabeculectomy or after phacoemulsification, IOL implantation and trabeculectomy in 1 or 2 steps.

BACKGROUND: To assess endothelial cell damage after glaucoma surgery and combined glaucoma and cataract surgery in one or two steps using confocal biomicroscopy. METHODS: This is an observational retrospective study. Eighty eyes from 62 patients between 60 and 83 years of age were studied. Eyes fell into a control group (n = 21) and three experimental groups, in which trabeculectomy (group 1; n = 21) or trabeculectomy and phacoemulsification and intraocular lens implantation had been performed, in one (group 2; n = 21) or two (group 3; n = 17) steps between 6 months and 5 years before. RESULTS: In the control group, mean corneal endothelial cell density (+/-SD) was 2,619 +/- 319 cells/mm(2), whereas in the experimental groups 1, 2 and 3 it was 2,447 +/- 425, 1,968 +/- 342 and 1,551 +/- 323 cells/mm(2) respectively. Cell densities found in the combined surgery groups were significantly smaller than the densities of the control or trabeculectomy groups. The variation coefficient of the endothelial cell area (+/-SD) was 41.19 +/- 7.46% in the control group and 38.9 +/- 6.02, 42.37 +/- 9.53 and 45.71 +/- 11.96% in groups 1, 2 and 3 respectively, differences that were not statistically significant. The percentage of hexagonality (+/-SD) was 51.10 +/- 8.41% in the control group and 51.4 +/- 6.88, 45.13 +/- 8.40 and 42.37 +/- 9.53% in the experimental groups 1, 2 and 3 respectively, but again there were no significant differences between them. CONCLUSIONS: Combined trabeculectomy, phacoemulsification and intraocular lens implantation causes more endothelial cell damage than trabeculectomy alone, and the two-step combined procedure causes more damage to the endothelium than the one-step combined procedure.

Coordinated Intervention of Microglial and Müller Cells in Light-Induced Retinal Degeneration.

Purpose: To analyze the role of microglial and Müller cells in the formation of rings of photoreceptor degeneration caused by phototoxicity. Methods: Two-month-old Sprague-Dawley rats were exposed to light and processed 1, 2, or 3 months later. Retinas were dissected as whole-mounts, immunodetected for microglial cells, Müller cells, and S- and L/M-cones and analyzed using fluorescence, thunder imaging, and confocal microscopy. Cone populations were automatically counted and isodensity maps constructed to document cone topography. Results: Phototoxicity causes a significant progressive loss of S- and L/M-cones of up to 68% and 44%, respectively, at 3 months after light exposure (ALE). One month ALE, we observed rings of cone degeneration in the photosensitive area of the superior retina. Two and 3 months ALE, these rings had extended to the central and inferior retina. Within the rings of cone degeneration, there were degenerating cones, often activated microglial cells, and numerous radially oriented processes of Müller cells that showed increased expression of intermediate filaments. Between 1 and 3 months ALE, the rings coalesced, and at the same time the microglial cells resumed a mosaic-like distribution, and there was a decrease of Müller cell gliosis at the areas devoid of cones. Conclusions: Light-induced photoreceptor degeneration proceeds with rings of cone degeneration, as observed in inherited retinal degenerations in which cone death is secondary to rod degeneration. The spatiotemporal relationship of cone death microglial cell activation and Müller cell gliosis within the rings of cone degeneration suggests that, although both glial cells are involved in the formation of the rings, they may have coordinated actions and, while microglial cells may be more involved in photoreceptor phagocytosis, Müller cells may be more involved in cone and microglial cell migration, retinal remodeling and glial seal formation.

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.

Short and long term axotomy-induced ERG changes in albino and pigmented rats.

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.