Short-term high-fat feeding exacerbates degeneration in retinitis pigmentosa by promoting retinal oxidative stress and inflammation.

A high-fat diet (HFD) can induce hyperglycemia and metabolic syndromes that, in turn, can trigger visual impairment. To evaluate the acute effects of HFD feeding on retinal degeneration, we assessed retinal function and morphology, inflammatory state, oxidative stress, and gut microbiome in dystrophic retinal degeneration 10 (rd10) mice, a model of retinitis pigmentosa, fed an HFD for 2 to 3 wk. Short-term HFD feeding impaired retinal responsiveness and visual acuity and enhanced photoreceptor degeneration, microglial cell activation, and Müller cell gliosis. HFD consumption also triggered the expression of inflammatory and oxidative markers in rd10 retinas. Finally, an HFD caused gut microbiome dysbiosis, increasing the abundance of potentially proinflammatory bacteria. Thus, HFD feeding drives the pathological processes of retinal degeneration by promoting oxidative stress and activating inflammatory-related pathways. Our findings suggest that consumption of an HFD could accelerate the progression of the disease in patients with retinal degenerative disorders.

Ischemia-Reperfusion Increases TRPM7 Expression in Mouse Retinas.

Ischemia is the main cause of cell death in retinal diseases such as vascular occlusions, diabetic retinopathy, glaucoma, or retinopathy of prematurity. Although excitotoxicity is considered the primary mechanism of cell death during an ischemic event, antagonists of glutamatergic receptors have been unsuccessful in clinical trials with patients suffering ischemia or stroke. Our main purpose was to analyze if the transient receptor potential channel 7 (TRPM7) could contribute to retinal dysfunction in retinal pathologies associated with ischemia. By using an experimental model of acute retinal ischemia, we analyzed the changes in retinal function by electroretinography and the changes in retinal morphology by optical coherence tomography (OCT) and OCT-angiography (OCTA). Immunohistochemistry was performed to assess the pattern of TRPM7 and its expression level in the retina. Our results show that ischemia elicited a decrease in retinal responsiveness to light stimuli along with reactive gliosis and a significant increase in the expression of TRPM7 in Müller cells. TRPM7 could emerge as a new drug target to be explored in retinal pathologies associated with ischemia.

Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy.

The purinergic receptor P2X7 (P2X7R) is implicated in all neurodegenerative diseases of the central nervous system. It is also involved in the retinal degeneration associated with glaucoma, age-related macular degeneration, and diabetic retinopathy, and its overexpression in the retina is evident in these disorders. Retinitis pigmentosa is a progressive degenerative disease that ultimately leads to blindness. Here, we investigated the expression of P2X7R during disease progression in the rd10 mouse model of RP. As the purinergic receptor P2X4 is widely co-expressed with P2X7R, we also studied its expression in the retina of rd10 mice. The expression of P2X7R and P2X4R was examined by immunohistochemistry, flow cytometry, and western blotting. In addition, we analyzed retinal functionality by electroretinographic recordings of visual responses and optomotor tests and retinal morphology. We found that the expression of P2X7R and P2X4R increased in rd10 mice concomitant with disease progression, but with different cellular localization. Our findings suggest that P2X7R and P2X4R might play an important role in RP progression, which should be further analyzed for the pharmacological treatment of inherited retinal dystrophies.

Dopaminergic Retinal Cell Loss and Visual Dysfunction in Parkinson Disease.

OBJECTIVE: Considering the demonstrated implication of the retina in Parkinson disease (PD) pathology and the importance of dopaminergic cells in this tissue, we aimed to analyze the state of the dopaminergic amacrine cells and some of their main postsynaptic neurons in the retina of PD. METHODS: Using immunohistochemistry and confocal microscopy, we evaluated morphology, number, and synaptic connections of dopaminergic cells and their postsynaptic cells, AII amacrine and melanopsin-containing retinal ganglion cells, in control and PD eyes from human donors. RESULTS: In PD, dopaminergic amacrine cell number was reduced between 58% and 26% in different retinal regions, involving a decline in the number of synaptic contacts with AII amacrine cells (by 60%) and melanopsin cells (by 35%). Despite losing their main synaptic input, AII cells were not reduced in number, but they showed cellular alterations compromising their adequate function: (1) a loss of mitochondria inside their lobular appendages, which may indicate an energetic failure; and (2) a loss of connexin 36, suggesting alterations in the AII coupling and in visual signal transmission from the rod pathway. INTERPRETATION: The dopaminergic system impairment and the affection of the rod pathway through the AII cells may explain and be partially responsible for the reduced contrast sensitivity or electroretinographic response described in PD. Also, dopamine reduction and the loss of synaptic contacts with melanopsin cells may contribute to the melanopsin retinal ganglion cell loss previously described and to the disturbances in circadian rhythm and sleep reported in PD patients. These data support the idea that the retina reproduces brain neurodegeneration and is highly involved in PD pathology. ANN NEUROL 2020;88:893-906.

Decrease in DHA and other fatty acids correlates with photoreceptor degeneration in retinitis pigmentosa.

Fatty acids, and especially docosahexaenoic acid (DHA), are essential for photoreceptor cell integrity and are involved in the phototransduction cascade. In this study, we analyzed the changes in the fatty acid profile in the retina of the rd10 mouse, model of retinitis pigmentosa, in order to identify potential risk factors for retinal degeneration and possible therapeutic approaches. Fatty acids from C57BL/6J and rd10 mouse retinas were extracted with Folch's method and analyzed by gas chromatography/mass spectrometry. Changes in retinal morphology were evaluated by immunohistochemistry. The rd10 mouse retina showed a decreased number of photoreceptor rows and alterations in photoreceptor morphology compared to C57BL/6J mice. The total amount of fatty acids dropped by 29.4% in the dystrophic retinas compared to C57BL/6J retinas. A positive correlation was found between the retinal content of specific fatty acids and the number of photoreceptor rows. We found that the amount of several short-chain and long-chain saturated fatty acids, as well as monounsaturated fatty acids, decreased in the retina of rd10 mice. Moreover, the content of the n-6 polyunsaturated fatty acid arachidonic acid and the n-3 polyunsaturated DHA decreased markedly in the dystrophic retina. The fall of DHA was more pronounced, hence the n-6/n-3 ratio was significantly increased in the diseased retina. The content of specific fatty acids in the retina decreased with photoreceptor degeneration in retinitis pigmentosa mice, with a remarkable reduction in DHA and other saturated and unsaturated fatty acids. These fatty acids could be essential for photoreceptor cell viability, and they should be evaluated for the design of therapeutical strategies and nutritional supplements.

New Nrf2-Inducer Compound ITH12674 Slows the Progression of Retinitis Pigmentosa in the Mouse Model rd10.

BACKGROUND/AIMS: It is well established that oxidative stress and inflammation are common pathogenic features of retinal degenerative diseases. ITH12674 is a novel compound that induces the transcription factor Nrf2; in so doing, the molecule exhibits anti-inflammatory, and antioxidant properties, and affords neuroprotection in rat cortical neurons subjected to oxidative stress. We here tested the hypothesis that ITH12674 could slow the retinal degeneration that causes blindness in rd10 mice, a model of retinitis pigmentosa. METHODS: Animals were intraperitoneally treated with 1 or 10 mg/Kg ITH12674 or placebo from P16 to P30. At P30, retinal functionality and visual acuity were analyzed by electroretinography and optomotor test. By immunohistochemistry we quantified the photoreceptor rows and analyzed their morphology and connectivity. Oxidative stress and inflammatory state was studied by Western blot, and microglia reactivity was monitored by flow cytometry. The blood-brain barrier permeation of ITH12674 was evaluated using a PAMPA-BBB assay. RESULTS: In rd10 mice treated with 10 mg/Kg of the compound, the following changes were observed (with respect to placebo): (i) a decrease of vision loss with higher scotopic a- and b-waves; (ii) increased visual acuity; (iii) preservation of cone photoreceptors morphology, as well as their synaptic connectivity; (iv) reduced expression of TNF-α and NF-κB; (v) increased expression of p38 MAPK and Atg12-Atg5 complex; and (vi) decreased CD11c, MHC class II and CD169 positive cell populations. CONCLUSION: These data support the view that a Nrf2 inducer compound may arise as a new therapeutic strategy to combat retinal neurodegeneration. At present, we are chemically optimising compound ITH12674 with the focus on improving its neuroprotective potential in retinal neurodegenerative diseases.

Cannabinoid-mediated retinal rescue correlates with improved circadian parameters in retinal dystrophic rats.

Ocular pathologies and blindness have been linked to circadian disorders. In previous studies, our group has demonstrated that retinitis pigmentosa is associated with degenerative changes in the melanopsin system and weaker circadian patterns. We have also shown that cannabinoids preserve retinal structure and function in dystrophic P23H rats. This study is consequently aimed at examining whether the morphologic and functional rescue of retinal degeneration by cannabinoids is associated with amelioration of circadian parameters. The synthetic cannabinoid HU210 (100 µg/kg, i.p.) or vehicle were administered to transgenic P23H rats three times per week, from postnatal day 24-90. Sprague-Dawley rats were used as a healthy control group. Locomotor activity and scotopic electroretinograms were recorded, and the retinal structure was analyzed at the end of the experiment. The ERG a- and b-wave amplitudes and photoreceptor cell number were more deteriorated in vehicle-administered P23H rats as compared to P23H rats treated with HU210. In cannabinoid-administered P23H rats, the locomotor activity circadian rhythms showed less disturbance than that observed in vehicle-administered P23H rats, the latter showing lower values for mesor, amplitude, acrophase, percentage of variance and non-parametric variables. A positive linear correlation was found between retinal values and circadian parameters of locomotor activity from P23H rats. This study thus provides evidence of a positive correlation between cannabinoid-mediated rescue of retinal structure and function and improvement of circadian rhythmicity.

Photosensitive Melanopsin-Containing Retinal Ganglion Cells in Health and Disease: Implications for Circadian Rhythms.

Melanopsin-containing retinal ganglion cells (mRGCs) represent a third class of retinal photoreceptors involved in regulating the pupillary light reflex and circadian photoentrainment, among other things. The functional integrity of the circadian system and melanopsin cells is an essential component of well-being and health, being both impaired in aging and disease. Here we review evidence of melanopsin-expressing cell alterations in aging and neurodegenerative diseases and their correlation with the development of circadian rhythm disorders. In healthy humans, the average density of melanopsin-positive cells falls after age 70, accompanied by age-dependent atrophy of dendritic arborization. In addition to aging, inner and outer retinal diseases also involve progressive deterioration and loss of mRGCs that positively correlates with progressive alterations in circadian rhythms. Among others, mRGC number and plexus complexity are impaired in Parkinson's disease patients; changes that may explain sleep and circadian rhythm disorders in this pathology. The key role of mRGCs in circadian photoentrainment and their loss in age and disease endorse the importance of eye care, even if vision is lost, to preserve melanopsin ganglion cells and their essential functions in the maintenance of an adequate quality of life.

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.

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.

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.

Neuroprotective effects of the cannabinoid agonist HU210 on retinal degeneration.

Cannabinoids have been demonstrated to exert neuroprotective effects on different types of neuronal insults. Here we have addressed the therapeutic potential of the synthetic cannabinoid HU210 on photoreceptor degeneration, synaptic connectivity and functional activity of the retina in the transgenic P23H rat, an animal model for autosomal dominant retinitis pigmentosa (RP). In P23H rats administered with HU210 (100 µg/kg, i.p.) from P24 to P90, ERG recordings showed an amelioration of vision loss, as compared to vehicle-administered animals. Under scotopic conditions, the maximum a-wave amplitudes recorded at P60 and P90 were higher in HU210-treated animals, as compared to the values obtained in untreated animals. The scotopic b-waves were significantly higher in treated animals than in untreated rats at P30, P60 and P90. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. HU210-treated animals had 40% more photoreceptors than untreated animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were also preserved in HU210-treated P23H rats. These results indicate that HU210 preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in P23H rats. These data suggest that cannabinoids are potentially useful to delay retinal degeneration in RP patients.

Non-haematopoietic Sca-1+ Cells in the Retina of Adult Mice Express Functional TLR2.

The mammal retina does not have the capacity to regenerate throughout life, although some stem and progenitor cells persist in the adult retina and might retain multipotentiality, as previously described in many tissues. In this work we demonstrate the presence of a small lineage- Sca-1+ cell population in the adult mouse retina which expresses functional TLR2 receptors as in vitro challenge with the pure TLR2 agonist Pam3CSK4 increases cell number and upregulates TLR2. Therefore, this population could be of interest in neuroregeneration studies to elucidate its role in these processes.

Characterization of a new murine retinal cell line (MU-PH1) with glial, progenitor and photoreceptor characteristics.

Unlike fish and amphibians, mammals do not regenerate retinal neurons throughout life. However, neurogenic potential may be conserved in adult mammal retina and it is necessary to identify the factors that regulate retinal progenitor cells (RPC) proliferative capacity to scope their therapeutic potential. Müller cells can be progenitors for retinal neuronal cells and can play an essential role in the restoration of visual function after retinal injury. Some members of the Toll-like receptor (TLR) family, TLR2, TLR3 and TLR4, are related to progenitor cells proliferation. Müller cells are important in retinal regeneration and stable cell lines are useful for the study of retinal stem cell biology. Our purpose was to obtain a Müller-derived cell line with progenitor characteristics and potential interest in regeneration processes. We obtained and characterized a murine Müller-derived cell line (MU-PH1), which proliferates indefinitely in vitro. Our results show that (i) MU-PH1 cells expresses the Müller cell markers Vimentin, S-100, glutamine synthetase and the progenitor and stem cell markers Nestin, Abcg2, Ascl1, α-tubulin and ß-III-tubulin, whereas lacks the expression of CRALBP, GFAP, Chx10, Pax6 and Notch1 markers; (ii) MU-PH1 cell line stably express the photoreceptor markers recoverin, transducin, rhodopsin, blue and red/green opsins and also melanopsin; (iii) the presence of opsins was confirmed by the recording of intracellular free calcium levels during light stimulation; (iv) MU-PH1 cell line also expresses the melatonin MT1 and MT2 receptors; (v) MU-PH1 cells express TLR1, 2, 4 and 6 mRNA; (vi) MU-PH1 express TLR2 at cell surface level; (vii) Candida albicans increases TLR2 and TLR6 mRNA expression; (viii) C. albicans or TLR selective agonists (Pam(3)CysSK(4), LPS) did not elicit morphological changes nor TNF-α secretion; (ix) C. albicans and Pam(3)CysSK(4) augmented MU-PH1 neurospheres formation in a statistically significant manner. Our results indicate that MU-PH1 cell line could be of great interest both as a photoreceptor model and in retinal regeneration approaches and that TLR2 may also play a role in retinal cell proliferation.

Starburst amacrine cells, involved in visual motion perception, loose their synaptic input from dopaminergic amacrine cells and degenerate in Parkinson's disease patients.

BACKGROUND: The main clinical symptoms characteristic of Parkinson's disease (PD) are bradykinesia, tremor, and other motor deficits. However, non-motor symptoms, such as visual disturbances, can be identified at early stages of the disease. One of these symptoms is the impairment of visual motion perception. Hence, we sought to determine if the starburst amacrine cells, which are the main cellular type involved in motion direction selectivity, are degenerated in PD and if the dopaminergic system is related to this degeneration. METHODS: Human eyes from control (n = 10) and PD (n = 9) donors were available for this study. Using immunohistochemistry and confocal microscopy, we quantified starburst amacrine cell density (choline acetyltransferase [ChAT]-positive cells) and the relationship between these cells and dopaminergic amacrine cells (tyrosine hydroxylase-positive cells and vesicular monoamine transporter-2-positive presynapses) in cross-sections and wholemount retinas. RESULTS: First, we found two different ChAT amacrine populations in the human retina that presented different ChAT immunoreactivity intensity and different expression of calcium-binding proteins. Both populations are affected in PD and their density is reduced compared to controls. Also, we report, for the first time, synaptic contacts between dopaminergic amacrine cells and ChAT-positive cells in the human retina. We found that, in PD retinas, there is a reduction of the dopaminergic synaptic contacts into ChAT cells. CONCLUSIONS: Taken together, this work indicates degeneration of starburst amacrine cells in PD related to dopaminergic degeneration and that dopaminergic amacrine cells could modulate the function of starburst amacrine cells. Since motion perception circuitries are affected in PD, their assessment using visual tests could provide new insights into the diagnosis of PD.

Prph2 knock-in mice recapitulate human central areolar choroidal dystrophy retinal degeneration and exhibit aberrant synaptic remodeling and microglial activation.

Central areolar choroidal dystrophy is an inherited disorder characterized by progressive choriocapillaris atrophy and retinal degeneration and is usually associated with mutations in the PRPH2 gene. We aimed to generate and characterize a mouse model with the p.Arg195Leu mutation previously described in patients. Heterozygous (Prph2WT/KI) and homozygous (Prph2KI/KI) mice were generated using the CRISPR/Cas9 system to introduce the p.Arg195Leu mutation. Retinal function was assessed by electroretinography and optomotor tests at 1, 3, 6, 9, 12, and 20 months of age. The structural integrity of the retinas was evaluated at the same ages using optical coherence tomography. Immunofluorescence and transmission electron microscopy images of the retina were also analyzed. Genetic sequencing confirmed that both Prph2WT/KI and Prph2KI/KI mice presented the p.Arg195Leu mutation. A progressive loss of retinal function was found in both mutant groups, with significantly reduced visual acuity from 3 months of age in Prph2KI/KI mice and from 6 months of age in Prph2WT/KI mice. Decreased amplitudes in the electroretinography responses were observed from 1 month of age in Prph2KI/KI mice and from 6 months of age in Prph2WT/KI mice. Morphological analysis of the retinas correlated with functional findings, showing a progressive decrease in retinal thickness of mutant mice, with earlier and more severe changes in the homozygous mutant mice. We corroborated the alteration of the outer segment structure, and we found changes in the synaptic connectivity in the outer plexiform layer as well as gliosis and signs of microglial activation. The new Prph2WT/KI and Prph2KI/KI murine models show a pattern of retinal degeneration similar to that described in human patients with central areolar choroidal dystrophy and appear to be good models to study the mechanisms involved in the onset and progression of the disease, as well as to test the efficacy of new therapeutic strategies.

Microglia activation and neuronal alterations in retinas from COVID-19 patients: correlation with clinical parameters.

BACKGROUND: Different ocular alterations have been described in patients with coronavirus disease 2019 (COVID-19). Our aim was to determine whether COVID-19 affected retinal cells and establish correlations with clinical parameters. METHODS: Retinal sections and flat-mount retinas from human donors with COVID-19 (n = 16) and controls (n = 15) were immunostained. The location of angiotensin-converting enzyme 2 (ACE2) and the morphology of microglial cells, Müller cells, astrocytes, and photoreceptors were analyzed by confocal microscopy. Microglial quantification and the area occupied by them were measured. Correlations among retinal and clinical parameters were calculated. RESULTS: ACE2 was mainly located in the Müller cells, outer segment of cones and retinal pigment epithelium. Cell bodies of Müller cells in COVID-19 group showed greater staining of ACE2 and cellular retinaldehyde-binding protein (CRALBP). The 81.3% of COVID-19 patients presented disorganization of honeycomb-like pattern formed by Müller cells. Gliosis was detected in 56.3% of COVID-19 patients compared to controls (40%) as well as epiretinal membranes (ERMs) or astrocytes protruding (50%). Activated or ameboid-shape microglia was the main sign in the COVID-19 group (93.8%). Microglial migration towards the vessels was greater in the COVID-19 retinas (P < 0.05) and the area occupied by microglia was also reduced (P < 0.01) compared to control group. Cone degeneration was more severe in the COVID-19 group. Duration of the disease, age and respiratory failure were the most relevant clinical data in relation with retinal degeneration. CONCLUSIONS: The retinas of patients with COVID-19 exhibit glial activation and neuronal alterations, mostly related to the inflammation, hypoxic conditions, and age.

Neuroprotective Effects of Tauroursodeoxicholic Acid Involves Vascular and Glial Changes in Retinitis Pigmentosa Model.

Purpose: Retinitis pigmentosa is primarily characterized by a massive photoreceptor loss. But a global retinal remodeling occurs in later stages of the disease. At that phase, glial cells and retinal vasculature are also strongly affected. The main aim of the present work is to assess if the bile acid Tauroursodeoxicholic acid (TUDCA), which has a demonstrated neuroprotective effect in numerous neurodegenerative diseases, is able to prevent glial and vascular degeneration in the P23H rat retina. Methods: Homozygous P23H (line 3) animals were injected weekly with a TUDCA (500 mg/kg, i.p.) or vehicle solution, from the postnatal day (P) 21 to P120. Sprague-Dawley rats (SD) were used as control. Retinal cross-sections and wholemounts were immunostained using different glial and vascular markers and visualized with confocal microscopy. Retinal blood vessels were stained with nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry and retinal vascular networks were drawn by hand using a camera lucida. Results: At P120, the photoreceptor degeneration observed in P23H rats was accompanied by a reduction in the vascular network density and complexity at the deep capillary plexus. In addition, astrocytes showed gliotic features and the outer processes of Müller cells displayed an aberrant distribution in ring-shaped structures. When treated with TUDCA, P23H rats displayed better-preserved vessels and capillary loops in the deep capillary plexus which are associated with the partial preservation of photoreceptors. TUDCA treatment also increased the number of astrocytes and reduced the presence of Müller cell process clusters in the outer retina. Conclusion: This work suggests that, besides its neuroprotective effect on photoreceptor cells, TUDCA treatment also protects from vascular and glial degeneration, a fact that encourages the use of TUDCA as a powerful therapy for neurodegenerative diseases.

Combined drug triads for synergic neuroprotection in retinal degeneration.

This review focuses on retina degeneration occurring during glaucoma, age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinitis pigmentosa (RP), and on the potential therapeutic use of triads of repositioned medicines, addressed to distinct but complementary targets, to prevent, delay or stop retina cell death. Although myriad pathogenic mechanisms have been implicated in these disorders, common signaling pathways leading to apoptotic cell death to all of them, and to all neurodegenerative diseases are (i) calcium dyshomeostasis/excitotoxicity; (ii) oxidative stress/mitochondrial dysfunction, and (iii) neuroinflammation/P2X7 receptor activation. From a therapeutic point of view, it is relevant to consider the multitarget approach based on the use of combined medicines acting on complementary pathogenic mechanisms that has been highly successful in the treatment of chronic diseases such as cancer, AIDS, pain, hypertension, Parkinson's disease, cardiac failure, depression, or the epilepsies as the basic mechanisms of cell death do not differ between the different CNS degenerative diseases. We suggest the multi-target therapy approach could be more effective compared with single-drug treatments. Used at doses lower than standard, these triads may also be safer and more efficient. After the establishment of a proof-of-concept in animal models of retinal degeneration, potential successful preclinical trials of such combinations may eventually drive to test this concept in clinical trials in patients, first to evaluate the safety and efficacy of the drug combinations in humans and then their therapeutic advantages, if any, seeking the prevention and/or the delay of retina degeneration and blindness.

Sodium Hyaluronate-Induced Ocular Hypertension in Rats Damages the Direction-Selective Circuit and Inner/Outer Retinal Plexiform Layers.

Purpose: To assess the changes in retinal morphology in a rat model of chronic glaucoma induced by ocular hypertension. Methods: Intraocular pressure (IOP) was surgically increased through weekly injections of sodium hyaluronate (HYA) in the anterior eye chamber of the left eye of male Wistar rats, whereas the right eyes were sham operated (salt solution). During the 10-week experimental period, IOP was measured weekly with a rebound tonometer. Retinal cryosections were prepared for histological/immunohistochemical analysis and morphometry. Results: IOP was higher in HYA-treated eyes than in sham-operated eyes along the 10-week period, which was significant from the fourth to the nineth week. Ocular hypertension in HYA-treated eyes was associated with morphologic and morphometric changes in bipolar cells, ON-OFF direction-selective ganglion cells, ON/OFF starburst amacrine cells, and inner plexiform layer sublamina. Conclusions: Serial HYA treatment in the rat anterior eye chamber results in mild-to-moderate elevated and sustained IOP and ganglion cell death, which mimics most human open-angle glaucoma hallmarks. The reduced number of direction-selective ganglion cells and starburst amacrine cells accompanied by a deteriorated ON/OFF plexus in this glaucoma model could lend insight to the abnormalities in motion perception observed in patients with glaucoma.