Norgestrel, a Progesterone Analogue, Promotes Significant Long-Term Neuroprotection of Cone Photoreceptors in a Mouse Model of Retinal Disease

Purpose: Retinitis pigmentosa (RP) refers to a group of inherited blinding retinal diseases, whereby the death of mutated rod photoreceptors is followed closely by the death of cone photoreceptors. Cone cell death can be hugely debilitating as color/daytime vision becomes impaired. Thus, treatments that are effective against cone cell death are urgently needed. Our research has been working toward development of a neuroprotective treatment for RP. We have previously demonstrated significant neuroprotective properties of norgestrel, a progesterone analogue, in the mouse retina. The current study further investigates the potential of norgestrel as a treatment for RP, with a focus on long-term preservation of cone photoreceptors. Methods: Using the well-established rd10 mouse model of RP, we administered a norgestrel-supplemented diet at postnatal day (P)30, following widespread loss of rod photoreceptors and at the outset of cone degeneration. We subsequently assessed cone cell morphology and retinal function at P50, P60, and P80, using immunohistochemistry, electroretinograph recordings, and optomotor testing. Results: While cone cell degeneration was widespread in the untreated rd10 retina, we observed profound preservation of cone photoreceptor morphology in the norgestrel-treated mice for at least 50 days, out to P80. This was demonstrated by up to 28-fold more cone arrestin-positive photoreceptors. This protection transpired to functional preservation at all ages. Conclusions: This work presents norgestrel as an incredibly promising long-term neuroprotective compound for the treatment of RP. Crucially, norgestrel could be used in the mid-late stages of the disease to protect remaining cone cells and help preserve color/daytime vision.Supported by grants from Science Foundation Ireland (SFI 13/IA/1783), Fighting Blindness Ireland (FB13COT), Spanish Ministry of Economy and Competitiveness (MINECO-FEDER-BFU2015-67139-R), Generalitat Valenciana (Prometeo 2016/158), and Carlos III Institute (ISCIII RETICS-FEDER RD16/0008/0016)

Retinitis pigmentosa is associated with shifts in the gut microbiome

The gut microbiome is known to influence the pathogenesis and progression of neurodegenerative diseases. However, there has been relatively little focus upon the implications of the gut microbiome in retinal diseases such as retinitis pigmentosa (RP). Here, we investigated changes in gut microbiome composition linked to RP, by assessing both retinal degeneration and gut microbiome in the rd10 mouse model of RP as compared to control C57BL/6J mice. In rd10 mice, retinal responsiveness to flashlight stimuli and visual acuity were deteriorated with respect to observed in age-matched control mice. This functional decline in dystrophic animals was accompanied by photoreceptor loss, morphologic anomalies in photoreceptor cells and retinal reactive gliosis. Furthermore, 16S rRNA gene amplicon sequencing data showed a microbial gut dysbiosis with differences in alpha and beta diversity at the genera, species and amplicon sequence variants (ASV) levels between dystrophic and control mice. Remarkably, four fairly common ASV in healthy gut microbiome belonging to Rikenella spp., Muribaculaceace spp., Prevotellaceae UCG-001 spp., and Bacilli spp. were absent in the gut microbiome of retinal disease mice, while Bacteroides caecimuris was significantly enriched in mice with RP. The results indicate that retinal degenerative changes in RP are linked to relevant gut microbiome changes. The findings suggest that microbiome shifting could be considered as potential biomarker and therapeutic target for retinal degenerative diseases.This study was funded by the Spanish Ministry of Economy Industry and Competitiveness (MINECO-FEDER BFU2015-67139-R and RTI2018-094248-B-I00), Spanish Ministry of Science and Innovation (MICINN-FEDER PID2019-106230RB-I00), Instituto de Salud Carlos III co-financed by European Regional Development funds (RETICS-FEDER RD16/0008/0016), Asociación Retina Asturias (ASOCIACIONRETINA1-20I), FARPE-FUNDALUCE (FUNDALUCE18-01), Generalitat Valenciana (FEDER IDIFEDER/2017/064) and Alicante’s University (UAIND18-05A)

Gradual Increase in Environmental Light Intensity Induces Oxidative Stress and Inflammation and Accelerates Retinal Neurodegeneration

Purpose: Retinitis pigmentosa (RP) is a blinding neurodegenerative disease of the retina that can be affected by many factors. The present study aimed to analyze the effect of different environmental light intensities in rd10 mice retina. Methods: C57BL/6J and rd10 mice were bred and housed under three different environmental light intensities: scotopic (5 lux), mesopic (50 lux), and photopic (300 lux). Visual function was studied using electroretinography and optomotor testing. The structural and morphological integrity of the retinas was evaluated by optical coherence tomography imaging and immunohistochemistry. Additionally, inflammatory processes and oxidative stress markers were analyzed by flow cytometry and western blotting. Results: When the environmental light intensity was higher, retinal function decreased in rd10 mice and was accompanied by light-dependent photoreceptor loss, followed by morphological alterations, and synaptic connectivity loss. Moreover, light-dependent retinal degeneration was accompanied by an increased number of inflammatory cells, which became more activated and phagocytic, and by an exacerbated reactive gliosis. Furthermore, light-dependent increment in oxidative stress markers in rd10 mice retina pointed to a possible mechanism for light-induced photoreceptor degeneration. Conclusions: An increase in rd10 mice housing light intensity accelerates retinal degeneration, activating cell death, oxidative stress pathways, and inflammatory cells. Lighting intensity is a key factor in the progression of retinal degeneration, and standardized lighting conditions are advisable for proper analysis and interpretation of experimental results from RP animal models, and specifically from rd10 mice. Also, it can be hypothesized that light protection could be an option to slow down retinal degeneration in some cases of RP.Supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO-FEDER BFU2015-67139-R); Spanish Ministry of Education (FPU16/04114); Instituto de Salud Carlos III co-financed by European Regional Development funds (RETICS-FEDER RD16/0008/0016); and Asociación Retina Asturias, FARPE-FUNDALUCE, Generalitat Valenciana (PROMETEO/2016/158, ACIF/2016/055 and FEDER IDIFEDER/2017/064)

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.Supported by the Spanish Ministry of Science and Innovation (MICINN-FEDER PID2019-106230RB-I00), 2), Institute of Health Carlos III (General Subdirection of Networks and Cooperative Research Centers RETICs OFTARED 2012-2021) co-financed by the European Regional Development fund (RD16/0008/0016 [Alicante] and RD16/0008/0022 [Valencia]), Asociación Retina Asturias (ASOCIACIONRETINA1-20I), Generalitat Valenciana (IDIFEDER/2017/064), and the Research funds from both Department of Surgery (Ophthalmology Unit) and Cellular-Molecular Ophthalmobiology Group of the University of Valencia (Spain)

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.We acknowledge financial support from the Ministerio de Ciencia e Innovación (FEDER-PID 2019-106230RB-I00), Ministerio de Universidades (FPU16/04114, FPU-18/02964). Generalitat Valenciana-FEDER (IDIFEDER/2017/064, PROMETEO/2021/024), Es Retina Asturias (2019/00286/001). MARSALAS21-35, financiado por la Unión Europea-Next Generation EU

Inherited Retinal Dystrophies: Role of Oxidative Stress and Inflammation in Their Physiopathology and Therapeutic Implications

Inherited retinal dystrophies (IRDs) are a large group of genetically and clinically heterogeneous diseases characterized by the progressive degeneration of the retina, ultimately leading to loss of visual function. Oxidative stress and inflammation play fundamental roles in the physiopathology of these diseases. Photoreceptor cell death induces an inflammatory state in the retina. The activation of several molecular pathways triggers different cellular responses to injury, including the activation of microglia to eliminate debris and recruit inflammatory cells from circulation. Therapeutical options for IRDs are currently limited, although a small number of patients have been successfully treated by gene therapy. Many other therapeutic strategies are being pursued to mitigate the deleterious effects of IRDs associated with oxidative metabolism and/or inflammation, including inhibiting reactive oxygen species’ accumulation and inflammatory responses, and blocking autophagy. Several compounds are being tested in clinical trials, generating great expectations for their implementation. The present review discusses the main death mechanisms that occur in IRDs and the latest therapies that are under investigation.This research was funded by DGA group B08_17R: Investigación en Retina y Sistema Visual and Fondo Europeo de Desarrollo Regional (FEDER) funds: “Una manera de hacer Europa”, Ministerio de Ciencia e Innovación (FEDER-PID 2019-106230RB-I00), Instituto de Salud Carlos III (PI20/00740-FEDER, RETICS-FEDER RD16/0008/0016), Generalitat Valenciana-FEDER (IDIFEDER/2017/064, PROMETEO/2021/024), Ministerio de Universidades (FPU16/04114), Es Retina Asturias (2019/00286/001). The APC was funded by DGA group B08_17R: Investigación en Retina y Sistema Visual (FEDER)

A Slc38a8 Mouse Model of FHONDA Syndrome Faithfully Recapitulates the Visual Deficits of Albinism Without Pigmentation Defects

Purpose: We aimed to generate and phenotype a mouse model of foveal hypoplasia, optic nerve decussation defects, and anterior segment dysgenesis (FHONDA), a rare disease associated with mutations in Slc38a8 that causes severe visual alterations similar to albinism without affecting pigmentation. Methods: The FHONDA mouse model was generated with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology using an RNA guide targeting the Scl38a8 murine locus. The resulting mice were backcrossed to C57BL/6J. Melanin content was measured using spectrophotometry. Retinal cell architecture was analyzed through light and electron microscopy. Retinal projections to the brain were evaluated with anterograde labelling in embryos and adults. Visual function was assessed by electroretinography (ERG) and the optomotor test (OT). Results: From numerous Slc38a8 mouse mutant alleles generated, we selected one that encodes a truncated protein (p.196Pro*, equivalent to p.199Pro* in the human protein) closely resembling a mutant allele described in patients (p.200Gln*). Slc38a8 mutant mice exhibit wild-type eye and coat pigmentation with comparable melanin content. Subcellular abnormalities were observed in retinal pigment epithelium cells of Slc38a8 mutant mice. Anterograde labeling experiments of retinal projections in embryos and adults showed a reduction of ipsilateral fibers. Functional visual analyses revealed a decreased ERG response in scotopic conditions and a reduction of visual acuity in mutant mice measured by OT. Conclusions: Slc38a8 mutant mice recapitulate the phenotype of patients with FHONDA concerning their normal pigmentation and their abnormal visual system, in the latter being a hallmark of all types of albinism. These mice will be helpful in better understanding the pathophysiology of this genetic condition.Funded by the Spanish Ministry of Economy and Competitiveness under BIO2015-70978-R, the Spanish Ministry of Science and Innovation under RTI2018-101223-B-I00, CIBERER and Fundación Ramón Areces to L.M. Additionally, Spanish Ministry of Science and Innovation (FEDER-PID2019-106230RB-I00, 2019) and Generalitat Valenciana IDIFEDER/2017/064, 2017, PROMETEO/2021/024, 2021 supported the work of N.C. Funds from INSERM, Sorbonne Université, Retina France and Genespoir supported the work of A.R., as well as LabEx LIFESENSES (ANR-10-LABX-65) and IHU FOReSIGHT (ANR-18-IAHU-01) for the Institut de la Vision, a doctoral fellowship from the French Ministry of Education and Research to V.C

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.This research was funded by grants from the Spanish Ministry of the Economy and Competitiveness (RTI2018-094248-B-I00), Spanish Ministry of Science and Innovation co-financed by European Regional Development Fund (MICINN-FEDER PID2019-106230RB-I00), Instituto de Salud Carlos III co-financed by European Regional Development Fund (RETICS-FEDER-RD16/0008/0016), Asociación Retina Asturias (ASOCIACIONRETINA1-20I), and Generalitat Valenciana (PROMETEO/2021/024, IDIFEDER/2017/064), and by a grant (MARSALAS21-35) to L.V

Cellular and molecular alterations in neurons and glial cells in inherited retinal degeneration

Multiple gene mutations have been associated with inherited retinal dystrophies (IRDs). Despite the spectrum of phenotypes caused by the distinct mutations, IRDs display common physiopathology features. Cell death is accompanied by inflammation and oxidative stress. The vertebrate retina has several attributes that make this tissue vulnerable to oxidative and nitrosative imbalance. The high energy demands and active metabolism in retinal cells, as well as their continuous exposure to high oxygen levels and light-induced stress, reveal the importance of tightly regulated homeostatic processes to maintain retinal function, which are compromised in pathological conditions. In addition, the subsequent microglial activation and gliosis, which triggers the secretion of pro-inflammatory cytokines, chemokines, trophic factors, and other molecules, further worsen the degenerative process. As the disease evolves, retinal cells change their morphology and function. In disease stages where photoreceptors are lost, the remaining neurons of the retina to preserve their function seek out for new synaptic partners, which leads to a cascade of morphological alterations in retinal cells that results in a complete remodeling of the tissue. In this review, we describe important molecular and morphological changes in retinal cells that occur in response to oxidative stress and the inflammatory processes underlying IRDs.This research was funded by the DGA group B08_17R: Investigación en Retina y Sistema Visual and Fondo Europeo de Desarrollo Regional (FEDER) funds: “Una manera de hacer Europa”, Ministerio de Ciencia e Innovación (FEDER-PID 2019-106230RB-I00), Instituto de Salud Carlos III (PI20/00740-FEDER, RETICS-FEDER RD16/0008/0016), Generalitat Valenciana-FEDER (IDIFEDER/2017/064, PROMETEO/2021/024), Ministerio de Universidades (FPU16/04114), Es Retina Asturias (2019/00286/001). The APC was funded by the DGA group B08_17R: Investigación en Retina y Sistema Visual (FEDER)

The Absence of Toll-Like Receptor 4 Mildly Affects the Structure and Function in the Adult Mouse Retina

The innate immune Toll-like receptor (TLR) family plays essential roles in cell proliferation, survival and function of the central nervous system. However, the way in which TLRs contribute to the development and maintenance of proper retinal structure and function remains uncertain. In this work, we assess the effect of genetic TLR4 deletion on the morphology and function of the retina in mice. Visual acuity and retinal responsiveness were evaluated in TLR4 knockout and wild type C57BL/6J control mice by means of an optomotor test and electroretinography, respectively, from P20 to P360. Retinal structure was also analyzed in both strains using confocal and electron microscopy. ERG data showed impaired retinal responsiveness in TLR4 KO mice, in comparison to wild type animals. The amplitudes of the scotopic a-waves were less pronounced in TLR4-deficient mice than in wild-type animals from P30 to P360, and TLR4 KO mice presented scotopic b-wave amplitudes smaller than those of age-matched control mice at all ages studied (P20 to P360). Visual acuity was also relatively poorer in TLR4 KO as compared to C57BL/6J mice from P20 to P360, with significant differences at P30 and P60. Immunohistochemical analysis of retinal vertical sections showed no differences between TLR4 KO and C57BL/6J mice, in terms of either photoreceptor number or photoreceptor structure. Horizontal cells also demonstrated no morphological differences between TLR4 KO and wild-type mice. However, TLR4 KO mice exhibited a lower density of bipolar cells (15% less at P30) and thus fewer bipolar cell dendrites than the wild type control mouse, even though both confocal and electron microscopy images showed no morphologic abnormalities in the synaptic contacts between the photoreceptors and second order neurons. Microglial cell density was significantly lower (26% less at P30) in TLR4 KO mice as compared to wild-type control mice. These results suggest that TLR4 deletion causes functional alterations in terms of visual response and acuity, probably through the loss of bipolar cells and microglia, but this receptor is not essential for the processing of visual information in the retina.This research was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO-FEDER BFU2015-67139-R), Spanish Ministry of Education (FPU14/03166), Instituto de Salud Carlos III (RETICS-FEDER RD16/0008/0016), and the Generalitat Valenciana (PROMETEO/2016/158 and ACIF/2016/055)