Rd10 Mice Research Articles

Monitoring Visual Cortical Activities During Progressive Retinal Degeneration Using Functional Bioluminescence Imaging.

Mouse models of inherited retinal degenerative diseases such as retinitis pigmentosa are characterized by degeneration of photoreceptors, which hinders the generation of signal to be transmitted to the visual cortex. By monitoring Ca2+-bioluminescence neural activity, we quantified changes in visual cortical activities in response to visual stimuli in RD10 mice during progression of retinal degeneration, which correlated with progressive deteriorations of electro-retinography signal from the eyes. The number of active neurons in the visual cortex, the intensity of Ca2+-bioluminescence response, and neural activation parameter showed progressive deterioration during aging. Further, we correlated the thinning of retina as measured by Optical Coherence Tomography with the decrease in visual cortical activities as retinal degeneration progressed. The present study establishes Ca2+-bioluminescence monitoring as a longitudinal imaging modality to characterize activities in visual cortex of retinal degenerative disease models and therapeutic interventions.

Intraperitoneal chromophore injections delay early-onset and rapid retinal cone degeneration in a mouse model of Leber congenital amaurosis

Highly expressed in the retinal pigment epithelium (RPE), the RPE-specific 65-kDa (RPE65) enzyme is indispensable to generate 11-cis-retinal (11cRAL), a chromophore for rhodopsin and cone photopigments. RPE65 deficiency can lead to Leber congenital amaurosis type 2 (LCA2), in which the isomerization of photobleached all-trans-retinal into photosensitive 11cRAL is blocked, ultimately causing severe retinal dysfunction and degeneration. The related mouse models, which are constructed through gene knockout or caused by spontaneous mutations, morphologically present with early-onset and rapid retinal cone cells degeneration, including loss of short-wavelength-sensitive cone opsins (S-opsins) and mislocalization of medium-wavelength-sensitive cone opsins (M-opsins). Studies have shown that routine Rpe65 gene replacement therapy, mediated by an adeno-associated virus (AAV) vector, can restore RPE65 protein. However, AAV transfection and Rpe65 transgene expression require at least one to two weeks, and the treatment cannot fully block the early-onset cone degeneration. To determine the feasibility of delaying cone degeneration before gene therapy, we investigated the impact of 11cRAL treatment in an early-age LCA2 retinal degeneration 12 (rd12) mouse model. Similar to human patients, the mouse model carries a spontaneous mutation in the Rpe65 gene, which results in disrupted endogenous 11cRAL regeneration. We found that RPE65 deficiency did not notably affect rodent retinal vessels. Under red light illumination, the rd12 mice were intraperitoneally injected with exogenous 11cRAL from postnatal day (P) 14 to P21. Three days after the last injection, a notable recovery of retinal function was observed using scotopic and photopic electroretinograms. Using optical coherence tomography and histological analyses of the deficient retinas, we found changes in the thickness of the photoreceptor outer segment (OS); this change could be rescued by early 11cRAL treatment. In addition, the treatment notably preserved M- and S-opsins, both of which maintained appropriate localization inside cone cells, as shown by the wild-type mice. In contrast, the age-matched untreated rd12 mice were characterized by retinal S-opsin loss and M-opsin mislocalization from the photoreceptor OS to the inner segment, outer nuclear layer, or outer plexiform layer. Notably, 11cRAL treatment could not maintain retinal function for a long time. Ten days after the last injection, the rod and M-cone electroretinograms significantly decreased, and S-cone responses almost extinguished. Our findings suggest that early 11cRAL treatment is useful for restoring retinal function and rescuing morphology in the rd12 mouse model, and the early-onset and rapid cone degeneration can be delayed before gene therapy.

Quantitative proteomic analysis after neuroprotective MyD88 inhibition in the retinal degeneration 10 mouse.

Progressive photoreceptor death occurs in blinding diseases such as retinitis pigmentosa. Myeloid differentiation primary response protein 88 (MyD88) is a central adaptor protein for innate immune system Toll‐like receptors (TLR) and induces cytokine secretion during retinal disease. We recently demonstrated that inhibiting MyD88 in mouse models of retinal degeneration led to increased photoreceptor survival, which was associated with altered cytokines and increased neuroprotective microglia. However, the identity of additional molecular changes associated with MyD88 inhibitor‐induced neuroprotection is not known. In this study, we used isobaric tags for relative and absolute quantification (iTRAQ) labelling followed by LC‐MS/MS for quantitative proteomic analysis on the rd10 mouse model of retinal degeneration to identify protein pathways changed by MyD88 inhibition. Quantitative proteomics using iTRAQ LC‐MS/MS is a high‐throughput method ideal for providing insight into molecular pathways during disease and experimental treatments. Forty‐two proteins were differentially expressed in retinas from mice treated with MyD88 inhibitor compared with control. Notably, increased expression of multiple crystallins and chaperones that respond to cellular stress and have anti‐apoptotic properties was identified in the MyD88‐inhibited mice. These data suggest that inhibiting MyD88 enhances chaperone‐mediated retinal protection pathways. Therefore, this study provides insight into molecular events contributing to photoreceptor protection from modulating inflammation.

Oxidative Stress Resistance 1 Gene Therapy Retards Neurodegeneration in the rd1 Mutant Mouse Model of Retinopathy.

PurposeOxidative stress is a major factor underlying many neurodegenerative diseases. However, antioxidant therapy has had mixed results, possibly because of its indiscriminate activity. The purpose of our study was to determine if the human OXR1 (hOXR1) antioxidant regulatory gene could protect neurons from oxidative stress and delay photoreceptor cell death.MethodsThe cone-like 661W cell line was transfected to stably express the hOXR1 gene. Oxidative stress was induced by the addition of hydrogen peroxide (H2O2). Intracellular levels of reactive oxygen species (ROS), caspase cleavage, and cellular resistance to oxidative stress were determined and compared between the control and hOXR1 cells. For in vivo analysis, AAV8-hOXR1 was injected subretinally into the rd1 mouse model of retinal degeneration. Functional and structural integrity of the photoreceptors were assessed using electroretinography (ERG), histology, and immunofluorescence analysis.ResultsExpression of hOXR1 increased cellular resistance and reduced ROS levels and caspase cleavage in the 661W cell line after H2O2-induced oxidative stress. Subretinal injection of AAV8-hOXR1 in the rd1 mice improved their photoreceptor light response, expression and localization of photoreceptor-specific proteins, and delayed retinal degeneration.ConclusionsOur results suggest that OXR1 is a potential therapy candidate for retinal degeneration. Because OXR1 targets oxidative stress, a common feature of many retinal degenerative diseases, it should be of therapeutic value to multiple retinal degenerative diseases.

CGMP-PKG dependent transcriptome in normal and degenerating retinas: Novel insights into the retinitis pigmentosa pathology

Retinitis Pigmentosa represents a group of genetic disorders that cause progressive vision loss via degeneration of photoreceptors, but there is in principle no treatment available. For any therapy development, a deeper comprehension of the disease-leading mechanism(s) at the molecular level is needed. Here we focused on the cGMP-PKG system, which has been suggested to be a driver in several models of the disease. To gain insights in its downstream signaling we manipulated the cGMP-PKG system with the aid of organotypic retinal explant cultures from either a mouse-based disease model, i.e. the rd1 mouse, or its healthy wild-type counterpart (wt), by adding different types of cGMP analogues to either inhibit or activate PKG in retinal explants from rd1 and wt, respectively. An RNA sequencing was then performed to study the cGMP-PKG dependent transcriptome. Expression changes of gene sets related to specific pathways or functions, that fulfilled criteria involving that the changes should match PKG activation and inhibition, were determined via bioinformatics. The analyses highlighted that several gene sets linked to oxidative phosphorylation and mitochondrial pathways were regulated by this enzyme system. Specifically, the expression of such pathway components was upregulated in the rd1 treated with PKG inhibitor and downregulated in the wt with PKG activator treatment, suggesting that cGMP-PKG act as a negative regulator in this context. Downregulation of energy production pathways may thus play an integral part in the mechanism behind the degeneration for at least several RP mutations.

Wolfberry-derived zeaxanthin dipalmitate delays retinal degeneration in a mouse model of retinitis pigmentosa through modulating STAT3, CCL2 and MAPK pathways.

Retinitis pigmentosa (RP) is a group of inherited photoreceptor degeneration diseases that causes blindness without effective treatment. The pathogenesis of retinal degeneration involves mainly oxidative stress and inflammatory responses. Zeaxanthin dipalmitate (ZD), a wolfberry-derived carotenoid, has anti-inflammatory and anti-oxidative stress effects. Here we investigated whether these properties of ZD can delay the retinal degeneration in rd10 mice, a model of RP, and explored its underlying mechanism. One shot of ZD or control vehicle was intravitreally injected into rd10 mice on postnatal day 16 (P16). Retinal function and structure of rd10 mice were assessed at P25, when rods degenerate substantially, using a visual behavior test, multi-electrode-array recordings and immunostaining. Retinal pathogenic gene expression and regulation of signaling pathways by ZD were explored using transcriptome sequencing and western blotting. Our results showed that ZD treatment improved the visual behavior of rd10 mice and delayed the degeneration of retinal photoreceptors. It also improved the light responses of photoreceptors, bipolar cells and retinal ganglion cells. The expression of genes that are involved in inflammation, apoptosis and oxidative stress were up-regulated in rd10 mice, and were reduced by ZD. ZD further reduced the activation of two key factors, signal transducer and activator of transcription 3 and chemokine (C-C motif) ligand 2, down-regulated the expression of the inflammatory factor GFAP, and inhibited extracellular signal regulated protein kinases and P38, but not the JNK pathways. In conclusion, ZD delays the degeneration of the rd10 retina both morphologically and functionally. Its anti-inflammatory function is mediated primarily through the signal transducer and activator of transcription 3, chemokine (C-C motif) ligand 2 and MAPK pathways. Thus, ZD may serve as a potential clinical candidate to treat RP.

Characterization and allogeneic transplantation of a novel transgenic cone-rich donor mouse line

ObjectivesCone photoreceptor transplantation is a potential treatment for macular diseases. The optimal conditions for cone transplantation are poorly understood, partly because of the scarcity of cones in donor mice. To facilitate allogeneic cone photoreceptor transplantation studies in mice, we aimed to create and characterize a donor mouse model containing a cone-rich retina with a cone-specific enhanced green fluorescent protein (EGFP) reporter. MethodsWe generated OPN1LW-EGFP/NRL−/− mice by crossing NRL−/− and OPN1LW-EGFP mice. We characterized the anatomical phenotype of OPN1LW-EGFP/NRL−/− mice using multimodal confocal scanning laser ophthalmoscopy (cSLO) imaging, immunohistology, and transmission electron microscopy. We evaluated retinal function using electroretinography (ERG), including 465 and 525 nm chromatic stimuli. Retinal sheets and cell suspensions from OPN1LW-EGFP/NRL−/− mice were transplanted subretinally into immunodeficient Rd1 mice. ResultsOPN1LW-EGFP/NRL−/− retinas were enriched with OPN1LW-EGFP+ and S-opsin+ cone photoreceptors in a dorsal-ventral distribution gradient. Cone photoreceptors co-expressing OPNL1W-EGFP and S-opsin significantly increased in OPN1LW-EGFP/NRL−/− compared to OPN1LW-EGFP mice. Temporal dynamics of rosette formation in the OPN1LW-EGFP/NRL−/− were similar as the NRL−/− with peak formation at P15. Rosettes formed preferentially in the ventral retina. The outer retina in P35 OPN1LW-EGFP/NRL−/− was thinner than NRL−/− controls. The OPN1LW-EGFP/NRL−/− ERG response amplitudes to 465 nm stimulation were similar to, but to 535 nm stimulation were lower than, NRL−/− controls. Three months after transplantation, the suspension grafts showed greater macroscopic degradation than sheet grafts. Retinal sheet grafts from OPN1LW-EGFP/NRL−/− mice showed greater S-opsin + cone survival than suspension grafts from the same strain. ConclusionsOPN1LW-EGFP/NRL−/− retinae were enriched with S-opsin+ photoreceptors. Sustained expression of EGFP facilitated the longitudinal tracking of transplanted donor cells. Transplantation of cone-rich retinal grafts harvested prior to peak rosette formation survived and differentiated into cone photoreceptor subtypes. Photoreceptor sheet transplantation may promote greater macroscopic graft integrity and S-opsin+ cone survival than cell suspension transplantation, although the mechanism underlying this observation is unclear at present. This novel cone-rich reporter mouse strain may be useful to study the influence of graft structure on cone survival.

Tlr2 Gene Deletion Delays Retinal Degeneration in Two Genetically Distinct Mouse Models of Retinitis Pigmentosa.

Although considered a rare retinal dystrophy, retinitis pigmentosa (RP) is the primary cause of hereditary blindness. Given its diverse genetic etiology (>3000 mutations in >60 genes), there is an urgent need for novel treatments that target common features of the disease. TLR2 is a key activator of innate immune response. To examine its role in RP progression we characterized the expression profile of Tlr2 and its adaptor molecules and the consequences of Tlr2 deletion in two genetically distinct models of RP: Pde6brd10/rd10 (rd10) and RhoP23H/+ (P23H/+) mice. In both models, expression levels of Tlr2 and its adaptor molecules increased in parallel with those of the proinflammatory cytokine Il1b. In rd10 mice, deletion of a single Tlr2 allele had no effect on visual function, as evaluated by electroretinography. However, in both RP models, complete elimination of Tlr2 attenuated the loss of visual function and mitigated the loss of photoreceptor cell numbers. In Tlr2 null rd10 mice, we observed decreases in the total number of microglial cells, assessed by flow cytometry, and in the number of microglia infiltrating the photoreceptor layers. Together, these results point to TLR2 as a mutation-independent therapeutic target for RP.

Retinal neovascularization induced by mutant Vldlr gene inhibited in an inherited retinitis pigmentosa mouse model: an in-vivo study

To explore whether the retinal neovascularization (NV) in a genetic mutant mice model could be ameliorated in an inherited retinitis pigmentosa (RP) mouse, which would help to elucidate the possible mechanism and prevention of retinal NV diseases in clinic. The Vldlr -/- mice, the genetic mutant mouse model of retinal NV caused by the homozygous mutation of Vldlr gene, with the rd1 mice, the inherited RP mouse caused by homozygous mutation of Pde6b gene were bred. Intercrossing of the above two mice led to the birth of the F1 hybrids, further inbreeding of which gave birth to the F2 offspring. The ocular genotypes and phenotypes of the mice from all generations were examined, with the F2 offspring grouped according to the genotypes. The rd1 mice exhibited the RP phenotype of outer retinal degeneration and loss of retinal function. The Vldlr -/- mice exhibited the phenotype of retinal NV obviously shown by the fundus fluorescein angiography. The F1 hydrides, with the heterozygote genotype, exhibited no phenotypes of RP or retinal NV. The F2 offspring with homozygous genotypes were grouped into four subgroups. They were the F2-I mice with the wild-type Pde6b and Vldlr genes (Pde6b+/+ -Vldlr+/+ ), which had normal ocular phenotypes; the F2-II mice with homozygous mutant Vldlr gene (Pde6b+/+ -Vldlr-/- ), which exhibited the retinal NV phenotype; the F2-III mice with homozygous mutant Pde6b gene (Pde6b-/- -Vldlr+/+ ), which exhibited the RP phenotype. Specifically, the F2-IV mice with homozygous mutant Vldlr and Pde6b gene (Pde6b-/- -Vldlr-/- ) showed only the RP phenotype, without the signs of retinal NV. The retinal NV can be inhibited by the RP phenotype, which implies the role of a hyperoxic state in treating retinal NV diseases.

Retinoid X receptor activation promotes photoreceptor survival and modulates the inflammatory response in a mouse model of retinitis pigmentosa

Photoreceptor cell (PHR) death is a hallmark of most retinal neurodegenerative diseases, in which inflammation plays a critical role. Activation of retinoid X receptors (RXR) modulates and integrates multiple cell functions, and has beneficial effects in animal models of chronic inflammatory diseases. Nonetheless, the mechanisms involved and their role in retina neuroprotection are poorly understood. In this work we assessed whether RXR activation prevents inflammation and/or PHR death in retinitis pigmentosa, an inherited retina neurodegeneration, using as an ex vivo model, retinas from the rd1 mice, a murine model of this disease. We demonstrated that rd1 retinas had lower levels of RXR alpha isoform than their wt counterparts at early developmental times, whereas its distribution pattern remained similar. In mixed neuro-glial cultures obtained from either rd1 or wt retinas, both PHR and Müller glial cells (MGC) expressed RXRalpha, and RXR activation by its synthetic pan-agonist PA024 selectively increased mRNA levels of RXRgamma isoform. PA024 decreased PHR death in rd1 mixed cultures; it reduced the amount of non-viable neurons, delayed the onset of PHR apoptosis, and decreased Bax mRNA levels. PA024 also reduced MGC reactivity in vitro before and at the onset of degeneration, decreasing GFAP expression, increasing glutamine synthetase mRNA levels, and promoting the transcription of the anti-inflammatory cytokine, Il-10. These results suggest that RXR activation rescues rd1 PHR and decreases MGC reactivity, promoting an anti-inflammatory environment in the rd1 retina, thus supporting the potential of RXR agonists as pharmacological tools for treating retina degenerative diseases.

Rb deficiency induces p21cip1 expression and delays retinal degeneration in rd1 mice

Retinitis pigmentosa (RP) is a major cause of inherited blindness, and there is presently no cure for RP. Rd1 mouse is the most commonly used RP animal model. Re-expression of cell cycle proteins in post-mitotic neurons is considered an important mechanism of neurodegenerative diseases, including RP. The retinoblastoma tumor suppressor (Rb) is a major regulator of cell cycle progression, yet its role in rd1 mouse retina and related signaling pathways have never been analyzed. By crossing α-Cre, Rbf/f mice with rd1 mice, p21cip1−/− mice, Cdk1f/f mice and Cdk2f/f mice, we established multiple rd1 mouse models with deletions of Rb gene, Cdkn1a (p21cip1) gene, Cdk1 and Cdk2 gene in the retina. Cdk inhibitor CR8 was injected into the vitreous of rd1 mouse to investigate its effects on photoreceptor survival. Rb gene knockout (KO) induces cell death in excitatory retinal neurons (rods, rod bipolar and ganglions) and ectopic proliferation of retinal cells; but it paradoxically delays the rod death of rd1 mice, which is primarily mediated by the Cdk inhibitor Cdkn1a (p21cip1). Interestingly, p21cip1 protects the ectopic dividing rd1 rod cells by inhibiting Cdk1 and Cdk2. However, inhibiting Cdk1 and Cdk2 in rd1 mice with non-dividing rods only has limited and transient protective effects. Our data suggest that there is no ectopic division of rd1 rod cells, and RbKO induces ectopic division but delays the death of rd1 rod cells. This reveals the important protective role of Rb-p21cip1-Cdk axis in rd1 rod cells. P21cip1 is a potential target for future therapy of RP.

Tribbles homolog 3-mediated targeting the AKT/mTOR axis in mice with retinal degeneration

Various retinal degenerative disorders manifest in alterations of the AKT/mTOR axis. Despite this, consensus on the therapeutic targeting of mTOR in degenerating retinas has not yet been achieved. Therefore, we investigated the role of AKT/mTOR signaling in rd16 retinas, in which we restored the AKT/mTOR axis by genetic ablation of pseudokinase TRB3, known to inhibit phosphorylation of AKT and mTOR. First, we found that TRB3 ablation resulted in preservation of photoreceptor function in degenerating retinas. Then, we learned that the mTOR downstream cellular pathways involved in the homeostasis of photoreceptors were also reprogrammed in rd16 TRB3−/− retinas. Thus, the level of inactivated translational repressor p-4E-BP1 was significantly increased in these mice along with the restoration of translational rate. Moreover, in rd16 mice manifesting decline in p-mTOR at P15, we found elevated expression of Beclin-1 and ATG5 autophagy genes. Thus, these mice showed impaired autophagy flux measured as an increase in LC3 conversion and p62 accumulation. In addition, the RFP-EGFP-LC3 transgene expression in rd16 retinas resulted in statistically fewer numbers of red puncta in photoreceptors, suggesting impaired late autophagic vacuoles. In contrast, TRIB3 ablation in these mice resulted in improved autophagy flux. The restoration of translation rate and the boost in autophagosome formation occurred concomitantly with an increase in total Ub and rhodopsin protein levels and the elevation of E3 ligase Parkin1. We propose that TRB3 may retard retinal degeneration and be a promising therapeutic target to treat various retinal degenerative disorders.

Inhibition of Epigenetic Modifiers LSD1 and HDAC1 Blocks Rod Photoreceptor Death in Mouse Models of Retinitis Pigmentosa

Epigenetic modifiers are increasingly being investigated as potential therapeutics to modify and overcome disease phenotypes. Diseases of the nervous system present a particular problem as neurons are postmitotic and demonstrate relatively stable gene expression patterns and chromatin organization. We have explored the ability of epigenetic modifiers to prevent degeneration of rod photoreceptors in a mouse model of retinitis pigmentosa (RP), using rd10 mice of both sexes. The histone modification eraser enzymes lysine demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1) are known to have dramatic effects on the development of rod photoreceptors. In the RP mouse model, inhibitors of these enzymes blocked rod degeneration, preserved vision, and affected the expression of multiple genes including maintenance of rod-specific transcripts and downregulation of those involved in inflammation, gliosis, and cell death. The neuroprotective activity of LSD1 inhibitors includes two pathways. First, through targeting histone modifications, they increase accessibility of chromatin and upregulate neuroprotective genes, such as from the Wnt pathway. We propose that this process is going in rod photoreceptors. Second, through nonhistone targets, they inhibit transcription of inflammatory genes and inflammation. This process is going in microglia, and lack of inflammation keeps rod photoreceptors alive.SIGNIFICANCE STATEMENT Retinal degenerations are a leading cause of vision loss. RP is genetically very heterogeneous, and the multiple pathways leading to cell death are one reason for the slow progress in identifying suitable treatments for patients. Here we demonstrate that inhibition of LSD1and HDAC1 in a mouse model of RP leads to preservation of rod photoreceptors and visual function, retaining of expression of rod-specific genes, and with decreased inflammation, cell death, and Müller cell gliosis. We propose that these epigenetic inhibitors cause more open and accessible chromatin, allowing expression of neuroprotective genes. A second mechanism that allows rod photoreceptor survival is suppression of inflammation by epigenetic inhibitors in microglia. Manipulation of epigenetic modifiers is a new strategy to fight neurodegeneration in RP.

Nutraceutical Supplementation Ameliorates Visual Function, Retinal Degeneration, and Redox Status in rd10 Mice.

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies characterized by progressive degeneration of photoreceptor cells. Ocular redox status is altered in RP suggesting oxidative stress could contribute to their progression. In this study, we investigated the effect of a mixture of nutraceuticals with antioxidant properties (NUT) on retinal degeneration in rd10 mice, a model of RP. NUT was orally administered to rd10 mice from postnatal day (PD) 9 to PD18. At PD18 retinal function and morphology were examined by electroretinography (ERG) and histology including TUNEL assay, immunolabeling of microglia, Müller cells, and poly ADP ribose polymers. Retinal redox status was determined by measuring the activity of antioxidant enzymes and some oxidative stress markers. Gene expression of the cytokines IL-6, TNFα, and IL-1β was assessed by real-time PCR. NUT treatment delayed the loss of photoreceptors in rd10 mice partially preserving their electrical responses to light stimuli. Moreover, it ameliorated redox status and reduced inflammation including microglia activation, upregulation of cytokines, reactive gliosis, and PARP overactivation. NUT ameliorated retinal functionality and morphology at early stages of RP in rd10 mice. This formulation could be useful as a neuroprotective approach for patients with RP in the future.

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.

Retinal Pigment Epithelium Remodeling in Mouse Models of Retinitis Pigmentosa.

In retinitis pigmentosa (RP), one of many possible genetic mutations causes rod degeneration, followed by cone secondary death leading to blindness. Accumulating evidence indicates that rod death triggers multiple, non-cell-autonomous processes, which include oxidative stress and inflammation/immune responses, all contributing to cone demise. Inflammation relies on local microglia and recruitment of immune cells, reaching the retina through breakdowns of the inner blood retinal barrier (iBRB). Leakage in the inner retina vasculature suggests similarly altered outer BRB, formed by junctions between retinal pigment epithelium (RPE) cells, which are crucial for retinal homeostasis, immune response, and privilege. We investigated the RPE structural integrity in three models of RP (rd9, rd10, and Tvrm4 mice) by immunostaining for zonula occludens-1 (ZO-1), an essential regulatory component of tight junctions. Quantitative image analysis demonstrated discontinuities in ZO-1 profiles in all mutants, despite different degrees of photoreceptor loss. ZO-1 interruption zones corresponded to leakage of in vivo administered, fluorescent dextran through the choroid-RPE interface, demonstrating barrier dysfunction. Dexamethasone, administered to rd10 mice for rescuing cones, also rescued RPE structure. Thus, previously undetected, stereotyped abnormalities occur in the RPE of RP mice; pharmacological targeting of inflammation supports a feedback loop leading to simultaneous protection of cones and the RPE.

Photoperiod integration in C3H rd1 mice.

In mammals, the suprachiasmatic nuclei (SCN) constitute the main circadian clock, receiving input from the retina which allows synchronization of endogenous biological rhythms with the daily light/dark cycle. Over the year, the SCN encodes photoperiodic variations through duration of melatonin secretion, with abundant nocturnal levels in winter and lower levels in summer. Thus, light information is critical to regulate seasonal reproduction in many species and is part of the central photoperiodic integration. Since intrinsically photosensitive retinal ganglion cells (ipRGCs) are vital for circadian photoentrainment and other nonvisual functions, we studied the contribution of ipRGCs in photoperiod integration in C3H retinal degeneration 1 (rd1) mice. We assessed locomotor activity and melatonin secretion in mice exposed to short or long photoperiods. Our results showed that rd1 mice are still responsive to photoperiod variations in term of locomotor activity, melatonin secretion, and regulation of the reproductive axis. In addition, retinas of animals exposed to short photoperiod exhibit higher melanopsin labeling intensity compared with the long photoperiod condition, suggesting seasonal-dependent changes within this photoreceptive system. These results show that ipRGCs in rd1 mice can still measure photoperiod and suggest a key role of melanopsin cells in photoperiod integration and the regulation of seasonal physiology.

Newly Identified Chemicals Preserve Mitochondrial Capacity and Decelerate Loss of Photoreceptor Cells in Murine Retinal Degeneration Models.

Purpose: Metabolic stress and associated mitochondrial dysfunction are implicated in retinal degeneration irrespective of the underlying cause. We identified seven unique chemicals from a Chembridge DiverSET screen and tested their protection against photoreceptor cell death in cell- and animal-based approaches. Methods: Calcium overload (A23187) was triggered in 661W murine photoreceptor-derived cells, and changes in redox potential and real-time changes in cellular metabolism were assessed using the MTT and Seahorse Biosciences XF assay, respectively. Cheminformatics to compare structures, and biodistribution in the living pig eye aided in selection of the lead compound. In-situ, retinal organ cultures of rd1 mouse and S334ter-line-3 rat were tested, in-vivo the light-induced retinal degeneration in albino Balb/c mice was used, assessing photoreceptor cell numbers histologically. Results: Of the seven chemicals, six were protective against A23187- and IBMX-induced loss of mitochondrial capacity, as measured by viability and respirometry in 661W cells. Cheminformatic analyses identified a unique pharmacophore with 6 physico-chemical features based on two compounds (CB11 and CB12). The protective efficacy of CB11 was further shown by reducing photoreceptor cell loss in retinal explants from two retinitis pigmentosa rodent models. Using eye drops, CB11 targeting to the pig retina was confirmed. The same eye drops decreased photoreceptor cell loss in light-stressed Balb/c mice. Conclusions: New chemicals were identified that protect from mitochondrial damage and lead to improved mitochondrial function. Using ex-vivo and in-vivo models, CB11 decreased the loss of photoreceptor cells in murine models of retinal degeneration and may be effective as treatment for different retinal dystrophies.

BFGF and insulin lead to migration of Müller glia to photoreceptor layer in rd1 mouse retina

Müller glia can act as endogenous stem cells and regenerate the missing neurons in the injured or degenerating retina in lower vertebrates. However, mammalian Müller glia, although can sometimes express stem cell markers and specific neuronal proteins in response to injury or degeneration, do not differentiate into functional neurons. We asked whether bFGF and insulin would stimulate the Müller glia to migrate, proliferate and differentiate into photoreceptors in rd1 mouse. We administered single or repeated (two or three) intravitreal injections of basic fibroblast growth factor (bFGF;200 μg) and insulin (2 μg) in 2-week-old rd1 mice. Müller glia were checked for proliferation, migration and differentiation using immunostaining. A single injection resulted within 5 days in a decrease in the numbers of Müller glia in the inner nuclear layer (INL) and a corresponding increase in the outer nuclear layer (ONL). The total number of Müller glia in the INL and ONL was unaltered, suggesting that they did not proliferate, but migrated from INL to ONL. However, maintaining the Müller cells in the ONL for two weeks or longer required repeated injections of bFGF and insulin. Interestingly, all Müller cells in the ONL expressed chx10, a stem cell marker. We did not find any immunolabeling for rhodopsin, m-opsin or s-opsin in the Müller glia in the ONL.

Spatial and temporal resolution of optogenetically recovered vision in ChR2-transduced mouse retina

Objective. Retinal ganglion cells (RGCs) represent an attractive target in vision restoration strategies, because they undergo little degeneration compared to other retinal neurons. Here we investigated the temporal and spatial resolution in adult photoreceptor-degenerated (rd10) mouse retinas, where RGCs have been transduced with the optogenetic actuator channelrhodopsin-2 (ChR2). Approach. The RGC spiking activity was recorded continuously with a CMOS-based microelectrode array during a variety of photostimulation protocols. The temporal resolution was assessed through Gaussian white noise stimuli and evaluated using a linear-nonlinear-Poisson model. Spatial sensitivity was assessed upon photostimulation with single rectangular pulses stepped across the retina and upon stimulation with alternating gratings of different spatial frequencies. Spatial sensitivity was estimated using logistic regression or by evaluating the spiking activity of independent RGCs. Main results. The temporal resolution after photostimulation displayed an about ten times faster kinetics as compared to physiological filters in wild-type RGCs. The optimal spatial resolution estimated using the logistic regression model was 10 µm and 87 µm based on the population response. These values correspond to an equivalent acuity of 1.7 or 0.2 cycles per degree, which is better than expected from the size of RGCs’ optogenetic receptive fields. Significance. The high temporal and spatial resolution obtained by photostimulation of optogenetically transduced RGCs indicate that high acuity vision restoration may be obtained by photostimulation of appropriately modified RGCs in photoreceptor-degenerated retinas.