Electroretinographical and histological study of mouse retina after optic nerve section: a comparison between wild-type and retinal degeneration 1 mice

Background: Retinal ganglion cell death underlies the pathophysiology of neurodegenerative disorders such as glaucoma or optic nerve trauma. To assess the potential influence of photoreceptor degeneration on retinal ganglion cell survival, and to evaluate functionality, we took advantage of the optic nerve section mouse model. Methods: Surviving retinal ganglion cells were double-stained by exposing both superior colliculi to fluorogold, and by applying dextran-tetramethylrhodamine to the injured optic nerve stump. To assess retinal function in wild-type animals, electroretinograms were recorded on the injured eyes and compared with the contralateral. Similar labelling experiments were carried out on retinal degeneration 1 mice. Surviving retinal ganglion cells were counted 21 days after axotomy and compared with wild-type mice. No functional experiments were performed on retinal degeneration 1 animals because they do not develop normal electroretinographical responses. Results: A significant decrease in retinal ganglion cell density was observed 6 days after axotomy in the wild type. Functional studies revealed that, in scotopic conditions, axotomy induced a significant amplitude decrease in the positive scotopic threshold response component of the electroretinogram. Such decrease paralleled cell loss, suggesting it may be an appropriate technique to evaluate functionality. When comparing retinal ganglion cell densities in wild-type and retinal degeneration 1 mice, a significant greater survival was observed on the latter. Conclusions: After optic nerve section, electroretinographical recordings exhibited a progressive decrease in the amplitude of the positive scotopic threshold response wave, reflecting ganglion cell loss. Interestingly, rod degeneration seemed, at least initially, to protect from axotomy-driven damage.

Loss of Outer Retinal Neurons and Circuitry Alterations in the DBA/2J Mouse

Purpose. The DBA/2J mouse line develops essential iris atrophy, pigment dispersion, and glaucomatous age-related changes, including an increase of IOP, optic nerve atrophy, and retinal ganglion cell (RGC) death. The aim of this study was to evaluate possible morphological changes in the outer retina of the DBA/2J mouse concomitant with disease progression and aging, based on the reduction of both the a- and b-waves and photopic flicker ERGs in this mouse line. Methods. Vertically sectioned DBA/2J mice retinas were evaluated at 3, 8, and 16 months of age using photoreceptor, horizontal, and bipolar cell markers. Sixteen-month-old C57BL/6 mice retinas were used as controls. Results. The DBA/2J mice had outer retinal degeneration at all ages, with the most severe degeneration in the oldest retinas. At 3 months of age, the number of photoreceptor cells and the thickness of the OPL were reduced. In addition, there was a loss of horizontal and ON-bipolar cell processes. At 8 months of age, RGC degeneration occurred in patches, and in the outer retina overlying these patches, cone morphology was impaired with a reduction in size as well as loss of outer segments and growth of horizontal and bipolar cell processes into the outer nuclear layer. At 16 months of age, connectivity between photoreceptors and horizontal and bipolar cell processes overlying these patches was lost. Conclusions. Retinal degeneration in DBA/2J mice includes photoreceptor death, loss of bipolar and horizontal cell processes, and loss of synaptic contacts in an aging-dependent manner.

Microglia activation in a model of retinal degeneration and TUDCA neuroprotective effects

Background: Retinitis pigmentosa is a heterogeneous group of inherited neurodegenerative retinal disorders characterized by a progressive peripheral vision loss and night vision difficulties, subsequently leading to central vision impairment. Chronic microglia activation is associated with various neurodegenerative diseases including retinitis pigmentosa. The objective of this study was to quantify microglia activation in the retina of P23H rats, an animal model of retinitis pigmentosa, and to evaluate the therapeutic effects of TUDCA (tauroursodeoxycholic acid), which has been described as a neuroprotective compound. Methods: For this study, homozygous P23H line 3 and Sprague-Dawley (SD) rats were injected weekly with TUDCA (500 mg/kg, ip) or vehicle (saline) from 20 days to 4 months old. Vertical retinal sections and whole-mount retinas were immunostained for specific markers of microglial cells (anti-CD11b, anti-Iba1 and anti-MHC-II). Microglial cell morphology was analyzed and the number of retinal microglial was quantified. Results: Microglial cells in the SD rat retinas were arranged in regular mosaics homogenously distributed within the plexiform and ganglion cell layers. In the P23H rat retina, microglial cells increased in number in all layers compared with control SD rat retinas, preserving the regular mosaic distribution. In addition, a large number of amoeboid CD11b-positive cells were observed in the P23H rat retina, even in the subretinal space. Retinas of TUDCA-treated P23H animals exhibited lower microglial cell number in all layers and absence of microglial cells in the subretinal space. Conclusions: These results report novel TUDCA anti-inflammatory actions, with potential therapeutic implications for neurodegenerative diseases, including retinitis pigmentosa.

Cellular responses following retinal injuries and therapeutic approaches for neurodegenerative diseases

Retinal neurodegenerative diseases like age-related macular degeneration, glaucoma, diabetic retinopathy and retinitis pigmentosa each have a different etiology and pathogenesis. However, at the cellular and molecular level, the response to retinal injury is similar in all of them, and results in morphological and functional impairment of retinal cells. This retinal degeneration may be triggered by gene defects, increased intraocular pressure, high levels of blood glucose, other types of stress or aging, but they all frequently induce a set of cell signals that lead to well-established and similar morphological and functional changes, including controlled cell death and retinal remodeling. Interestingly, an inflammatory response, oxidative stress and activation of apoptotic pathways are common features in all these diseases. Furthermore, it is important to note the relevant role of glial cells, including astrocytes, Müller cells and microglia, because their response to injury is decisive for maintaining the health of the retina or its degeneration. Several therapeutic approaches have been developed to preserve retinal function or restore eyesight in pathological conditions. In this context, neuroprotective compounds, gene therapy, cell transplantation or artificial devices should be applied at the appropriate stage of retinal degeneration to obtain successful results. This review provides an overview of the common and distinctive features of retinal neurodegenerative diseases, including the molecular, anatomical and functional changes caused by the cellular response to damage, in order to establish appropriate treatments for these pathologies.

Phosphorylated α-synuclein-immunoreactive retinal neuronal elements in Parkinson's disease subjects

Visual symptoms are relatively common in Parkinson's disease (PD) and optical coherence tomography has indicated possible retinal thinning. Accumulation of aggregated α-synuclein is thought to be a central pathogenic event in the PD brain but there have not as yet been reports of retinal synucleinopathy. Retinal wholemounts were prepared from subjects with a primary clinicopathological diagnosis of PD (N = 9), dementia with Lewy bodies (DLB; N = 3), Alzheimer's disease (N = 3), progressive supranuclear palsy (N = 2) as well as elderly normal control subjects (N = 4). These were immunohistochemically stained with an antibody against α-synuclein phosphorylated at serine 129, which is a specific molecular marker of synucleinopathy. Phosphorylated α-synuclein-immunoreactive (p-syn IR) nerve fibers were present in 7/9 PD subjects and in 1/3 DLB subjects; these were sparsely distributed and superficially located near or at the inner retinal surface. The fibers were either long and straight or branching, often with multiple en-passant varicosities along their length. The straight fibers most often had an orientation that was radial with respect to the optic disk. Together, these features are suggestive of either retinopetal/centrifugal fibers or of ganglion cell axons. In one PD subject there were sparse p-syn IR neuronal cell bodies with dendritic morphology suggestive of G19 retinal ganglion cells or intrinsically photosensitive ganglion cells. There were no stained nerve fibers or other specific staining in any of the non-PD or non-DLB subjects. It is possible that at least some of the observed visual function impairments in PD subjects might be due to α-synucleinopathy.

Corneal Biomechanics, Retinal Nerve Fiber Layer, and Optic Disc in Children

Purpose: To evaluate the possible associations between corneal biomechanical parameters, optic disc morphology, and retinal nerve fiber layer (RNFL) thickness in healthy white Spanish children. Methods: This cross-sectional study included 100 myopic children and 99 emmetropic children as a control group, ranging in age from 6 to 17 years. The Ocular Response Analyzer was used to measure corneal hysteresis (CH) and corneal resistance factor. The optic disc morphology and RNFL thickness were assessed using posterior segment optical coherence tomography (Cirrus HD-OCT). The axial length was measured using an IOLMaster, whereas the central corneal thickness was measured by anterior segment optical coherence tomography (Visante OCT). Results: The mean (±SD) age and spherical equivalent were 12.11 (±2.76) years and −3.32 (±2.32) diopters for the myopic group and 11.88 (±2.97) years and +0.34 (±0.41) diopters for the emmetropic group. In a multivariable mixed-model analysis in myopic children, the average RNFL thickness and rim area correlated positively with CH (p = 0.007 and p = 0.001, respectively), whereas the average cup-to-disc area ratio correlated negatively with CH (p = 0.01). We did not observe correlation between RNFL thickness and axial length (p = 0.05). Corneal resistance factor was only positively correlated with the rim area (p = 0.001). The central corneal thickness did not correlate with the optic nerve parameters or with RNFL thickness. These associations were not found in the emmetropic group (p > 0.05 for all). Conclusions: The corneal biomechanics characterized with the Ocular Response Analyzer system are correlated with the optic disc profile and RNFL thickness in myopic children. Low CH values may indicate a reduction in the viscous dampening properties of the cornea and the sclera, especially in myopic children.

Natural Compounds from Saffron and Bear Bile Prevent Vision Loss and Retinal Degeneration

All retinal disorders, regardless of their aetiology, involve the activation of oxidative stress and apoptosis pathways. The administration of neuroprotective factors is crucial in all phases of the pathology, even when vision has been completely lost. The retina is one of the most susceptible tissues to reactive oxygen species damage. On the other hand, proper development and functioning of the retina requires a precise balance between the processes of proliferation, differentiation and programmed cell death. The life-or-death decision seems to be the result of a complex balance between pro- and anti-apoptotic signals. It has been recently shown the efficacy of natural products to slow retinal degenerative process through different pathways. In this review, we assess the neuroprotective effect of two compounds used in the ancient pharmacopoeia. On one hand, it has been demonstrated that administration of the saffron constituent safranal to P23H rats, an animal model of retinitis pigmentosa, preserves photoreceptor morphology and number, the capillary network and the visual response. On the other hand, it has been shown that systemic administration of tauroursodeoxycholic acid (TUDCA), the major component of bear bile, to P23H rats preserves cone and rod structure and function, together with their contact with postsynaptic neurons. The neuroprotective effects of safranal and TUDCA make these compounds potentially useful for therapeutic applications in retinal degenerative diseases.

Neuroprotective Effect of Tauroursodeoxycholic Acid on N-Methyl-D-Aspartate-Induced Retinal Ganglion Cell Degeneration

Retinal ganglion cell degeneration underlies the pathophysiology of diseases affecting the retina and optic nerve. Several studies have previously evidenced the anti-apoptotic properties of the bile constituent, tauroursodeoxycholic acid, in diverse models of photoreceptor degeneration. The aim of this study was to investigate the effects of systemic administration of tauroursodeoxycholic acid on N-methyl-D-aspartate (NMDA)-induced damage in the rat retina using a functional and morphological approach. Tauroursodeoxycholic acid was administered intraperitoneally before and after intravitreal injection of NMDA. Three days after insult, full-field electroretinograms showed reductions in the amplitudes of the positive and negative-scotopic threshold responses, scotopic a- and b-waves and oscillatory potentials. Quantitative morphological evaluation of whole-mount retinas demonstrated a reduction in the density of retinal ganglion cells. Systemic administration of tauroursodeoxycholic acid attenuated the functional impairment induced by NMDA, which correlated with a higher retinal ganglion cell density. Our findings sustain the efficacy of tauroursodeoxycholic acid administration in vivo, suggesting it would be a good candidate for the pharmacological treatment of degenerative diseases coursing with retinal ganglion cell loss.

Long time remodeling during retinal degeneration evaluated by optical coherence tomography, immunocytochemistry and fundus autofluorescence

Purpose: To characterize the relationship between fundus autofluorescence (FAF), Optical Coherence Tomography (OCT) and immunohistochemistry (IHC) over the course of chronic retinal degeneration in the P23H rat. Methods: Homozygous albino P23H rats, Sprague–Dawley (SD) rats as controls and pigmented Long Evans (LE) rats were used. A Spectralis HRA OCT system was used for scanning laser ophthalmoscopy (SLO) imaging OCT and angiography. To determine FAF, fluorescence was excited using diode laser at 488 nm. A fast retina map OCT was performed using the optic nerve as a landmark. IHC was performed to correlate with the findings of OCT and FAF changes. Results: During the course of retinal degeneration, the FAF pattern evolved from some spotting at 2 months old to a mosaic of hyperfluorescent dots in rats 6 months and older. Retinal thicknesses progressively diminished over the course of the disease. At later stages of degeneration, OCT documented changes in the retinal layers, however, IHC better identified the cell loss and remodeling changes. Angiography revealed attenuation of the retinal vascular plexus with time. Conclusion: We provide for the first time a detailed long-term analysis of the course of retinal degeneration in P23H rats using a combination of SLO and OCT imaging, angiography, FAF and IHC. Although, the application of noninvasive methods enables longitudinal studies and will decrease the number of animals needed for a study, IHC is still an essential tool to identify retinal changes at the cellular level.

Astrocytes and Müller Cell Alterations During Retinal Degeneration in a Transgenic Rat Model of Retinitis Pigmentosa

Purpose: Retinitis pigmentosa includes a group of progressive retinal degenerative diseases that affect the structure and function of photoreceptors. Secondarily to the loss of photoreceptors, there is a reduction in retinal vascularization, which seems to influence the cellular degenerative process. Retinal macroglial cells, astrocytes, and Müller cells provide support for retinal neurons and are fundamental for maintaining normal retinal function. The aim of this study was to investigate the evolution of macroglial changes during retinal degeneration in P23H rats. Methods: Homozygous P23H line-3 rats aged from P18 to 18 months were used to study the evolution of the disease, and SD rats were used as controls. Immunolabeling with antibodies against GFAP, vimentin, and transducin were used to visualize macroglial cells and cone photoreceptors. Results: In P23H rats, increased GFAP labeling in Müller cells was observed as an early indicator of retinal gliosis. At 4 and 12 months of age, the apical processes of Müller cells in P23H rats clustered in firework-like structures, which were associated with ring-like shaped areas of cone degeneration in the outer nuclear layer. These structures were not observed at 16 months of age. The number of astrocytes was higher in P23H rats than in the SD matched controls at 4 and 12 months of age, supporting the idea of astrocyte proliferation. As the disease progressed, astrocytes exhibited a deteriorated morphology and marked hypertrophy. The increase in the complexity of the astrocytic processes correlated with greater connexin 43 expression and higher density of connexin 43 immunoreactive puncta within the ganglion cell layer (GCL) of P23H vs. SD rat retinas. Conclusions: In the P23H rat model of retinitis pigmentosa, the loss of photoreceptors triggers major changes in the number and morphology of glial cells affecting the inner retina.

Correlation between SD-OCT, immunocytochemistry and functional findings in an animal model of retinal degeneration

Purpose: The P23H rhodopsin mutation is an autosomal dominant cause of retinitis pigmentosa (RP). The degeneration can be tracked using different anatomical and functional methods. In our case, we evaluated the anatomical changes using Spectral-Domain Optical Coherence Tomography (SD-OCT) and correlated the findings with retinal thickness values determined by immunocytochemistry.Methods: Pigmented rats heterozygous for the P23H mutation, with ages between P18 and P180 were studied. Function was assessed by means of optomotor testing and ERGs. Retinal thicknesses measurements, autofluorescence and fluorescein angiography were performed using Spectralis OCT. Retinas were studied by means of immunohistochemistry. Results: Between P30 and P180, visual acuity decreased from 0.500 to 0.182 cycles per degree (cyc/deg) and contrast sensitivity decreased from 54.56 to 2.98 for a spatial frequency of 0.089 cyc/deg. Only cone-driven b-wave responses reached developmental maturity. Flicker fusions were also comparable at P29 (42 Hz). Double flash-isolated rod-driven responses were already affected at P29. Photopic responses revealed deterioration after P29.A reduction in retinal thicknesses and morphological modifications were seen in OCT sections. Statistically significant differences were found in all evaluated thicknesses. Autofluorescence was seen in P23H rats as sparse dots. Immunocytochemistry showed a progressive decrease in the outer nuclear layer (ONL), and morphological changes. Although anatomical thickness measures were significantly lower than OCT values, there was a very strong correlation between the values measured by both techniques.Conclusions: In pigmented P23H rats, a progressive deterioration occurs in both retinal function and anatomy. Anatomical changes can be effectively evaluated using SD-OCT and immunocytochemistry, with a good correlation between their values, thus making SD-OCT an important tool for research in retinal degeneration.

SPECTRAL DOMAIN-OPTICAL COHERENCE TOMOGRAPHY IMAGE CONTRAST AND BACKGROUND COLOR SETTINGS INFLUENCE IDENTIFICATION OF RETINAL STRUCTURES.

PURPOSE To evaluate image contrast and color setting on assessment of retinal structures and morphology in spectral-domain optical coherence tomography. METHODS Two hundred and forty-eight Spectralis spectral-domain optical coherence tomography B-scans of 62 patients were analyzed by 4 readers. B-scans were extracted in 4 settings: W + N = white background with black image at normal contrast 9; W + H = white background with black image at maximum contrast 16; B + N = black background with white image at normal contrast 12; B + H = black background with white image at maximum contrast 16. Readers analyzed the images to identify morphologic features. Interreader correlation was calculated. Differences between Fleiss-kappa correlation coefficients were examined using bootstrap method. Any setting with significantly higher correlation coefficient was deemed superior for evaluating specific features. RESULTS Correlation coefficients differed among settings. No single setting was superior for all respective spectral-domain optical coherence tomography parameters (P = 0.3773). Some variables showed no differences among settings. Hard exudates and subretinal fluid were best seen with B + H (κ = 0.46, P = 0.0237 and κ = 0.78, P = 0.002). Microaneurysms were best seen with W + N (κ = 0.56, P = 0.025). Vitreomacular interface, enhanced transmission signal, and epiretinal membrane were best identified using all color/contrast settings together (κ = 0.44, P = 0.042, κ = 0.57, P = 0.01, and κ = 0.62, P ≤ 0.0001). CONCLUSION Contrast and background affect the evaluation of retinal structures on spectral-domain optical coherence tomography images. No single setting was superior for all features, though certain changes were best seen with specific settings.

Quantification of retinal layer thickness changes in acute macular neuroretinopathy.

PURPOSE To quantitatively evaluate retinal layer thickness changes in acute macular neuroretinopathy (AMN). METHODS AMN areas were identified using near-infrared reflectance (NIR) images. Intraretinal layer segmentation using Heidelberg software was performed. The inbuilt ETDRS -grid was moved onto the AMN lesion and the mean retinal layer thicknesses of the central grid were recorded and compared with the corresponding area of the fellow eye at initial presentation and during follow-up. RESULTS Eleven patients were included (mean age 26±6 years). AMN lesions at baseline had a significantly thinner outer nuclear layer (ONL) (51±21 µm vs 73±17 µm, p=0.002). The other layers, including inner nuclear layer (37±8 µm vs 38±6 µm, p=0.9) and outer plexiform layer (OPL) (45±19 µm vs 33±16 µm, p=0.1) did not show significant differences between the study eyes and fellow eyes. Adjacent to NIR image lesions, areas of OPL thickening were identified (study eye: 50±14 µm vs fellow eye: 39±16 µm, p=0.005) with corresponding thinning of ONL (study eye: 52±16 µm vs fellow eye: 69±16 µm, p=0.002). CONCLUSIONS AMN presents with characteristic quantitative retinal changes and the extent of the lesion may be more extensive than initially presumed from NIR image lesions.

Retinal ganglion cell layer change in patients treated with anti-VEGF for neovascular age related macular degeneration.

PURPOSE To evaluate macular retinal ganglion cell thickness in patients with neovascular age-related macular degeneration (AMD) and intravitreal anti-vascular endothelial growth factor (VEGF) therapy. DESIGN Retrospective case series with fellow-eye comparison METHODS: Patients with continuous unilateral anti-VEGF treatment for sub- and juxtafoveal neovascular AMD and a minimum follow-up of 24 months were included. The retinal nerve fiber (RNFL) and retinal ganglion cell layer (RGCL) in the macula were segmented using an ETDRS grid. RNFL and RGCL thickness of the outer ring of the ETDRS grid were quantified at baseline and after repeated anti-VEGF injections, and compared to the patients' untreated fellow eye. Furthermore, best-corrected visual acuity (BCVA), age, and retinal pigment epithelium (RPE) atrophy were recorded and correlated with RNFL and RGCL. RESULTS Sixty eight eyes of 34 patients (23 female and 11 male; mean age 76.7 (SD±8.2) with a mean number of 31.5 (SD ±9.8) anti-VEGF injections and a mean follow-up period of 45.3 months (SD±10.5) were included. Whereas the RGCL thickness decreased significantly compared to the non-injected fellow eye (p=0.01) the decrease of the RNFL was not significant. Visual acuity gain was significantly correlated with RGCL thickness (r=0.52, p<0.05) at follow-up and negatively correlated (r=-0.41, p<0.05) with age. Presence of RPE atrophy correlated negatively with the RGCL thickness at follow-up (r= -0.37, p=0.03). CONCLUSION During the course of long term anti-VEGF therapy there is a significant decrease of the RGCL in patients with neovascular AMD to the fellow (untreated) eye.