Retinal degenerations are associated with aberrant activation of inflammation. Interleukin-27 (IL-27) is a cytokine with anti-inflammatory and neuroprotective activities in the CNS. However, the molecular and cellular mechanisms by which IL-27 mediates its neuroprotective effects remain unclear. The rd10 mouse model of retinal degeneration received an intravitreal injection of IL-27 or saline prior to degeneration. IL-27 induced sustained retina protection for at least four weeks after injection. SnRNA-seq analysis demonstrated suppression of a unique Muller glia subpopulation in IL-27 treated mice. Moreover, snRNA-seq revealed differential expression of distinct gene categories across glial and photoreceptor cell types, including genes involved in ATP synthesis, metabolism, protein translation, apoptosis, inflammation and extracellular matrix. Therefore, reducing reactive inflammation in early retinal disease with IL-27 leads to changes in numerous cellular pathways and sustained neuroprotection in rd10 mice. Additionally, our findings suggest that suppression of a specific Muller glia subpopulation contributes to the neuroprotective effect of IL-27. Therefore, this study identified new pathways of neuroprotection in the retina.

Air pollution is a global environmental hazard and is associated with increased severity and progression of several retinal diseases, including glaucoma and age-related macular degeneration. Muller glia are radial glia in the retina that play essential roles in metabolic support, redox balance and neuroprotection. Despite their importance, the effects of pollution on retinal glial responses are not well characterized. The objectives of this study were to determine the effects of a test dust preparation on viability, injury responses and oxidative stress levels in human Muller glia cell line. Arizona test dust (ATD) contains silica, oxides, particulate matter smaller than 10 μm and other potentially harmful components. Subconfluent cultures of Muller glia MIO-M1 cells were exposed to ATD (0.1 - 50 μg/mL) and cell viability, reactive oxygen species (ROS) production, antioxidant gene expression and transcriptomic analyses were performed. ATD exposure led to increased ROS without reducing viability, and was accompanied by reduced expression of antioxidant genes NRF2 and SOD1. Bulk RNA-seq analysis demonstrated downregulation of numerous mitochondrial genes and alterations in genes regulating migration, inflammation and adhesion. Therefore, these findings indicate that ATD pollution disrupts antioxidant protective mechanisms and mitochondrial gene expression in Muller glia, potentially leading to oxidative imbalance and transcriptional alterations that could contribute to retinal dysfunction.

Air pollution is associated with increased incidence of age-related macular degeneration (AMD), glaucoma and other retinal diseases. The time course of molecular and cellular changes induced by chronic air pollution exposure that lead to retinal pathology are unknown. In this study, we investigated the effects of moderate levels of air pollution on visual acuity and retinal phenotypes in mice using Arizona test dust (ATD) as a surrogate for ambient air pollution. Mice were exposed to aerosolized standardized test dust for three hours a day, four days a week, for up to four months in a custom-built chamber. Controls were exposed to room air. Visual function was assessed using an optomotor assay and demonstrated reduced visual acuity after one month of exposure that persisted throughout the study. Rod and cone photoreceptors also showed temporarily decreased light-evoked responses that returned to normal levels by four months. Furthermore, reduced cone photoreceptors and retinal ganglion cells were observed whereas markers of retinal stress, including Iba1-positive microglia/macrophage and GFAP expression in macroglia, were not significantly elevated. Molecular analyses indicated elevated expression of genes in the Nrf2-ARE oxidative stress response pathway. Therefore, moderate levels of aerosolized ATD caused a persistent decline in visual acuity, mild retinal degeneration and transient functional changes. This study provides new information into the pathogenesis of pollution-induced retinal damage and establishes a new mouse model for investigating detrimental effects of moderate levels of air pollution in the retina.

Optic nerve (ON) injury leads to retinal ganglion cell (RGC) degeneration and axonal atrophy. Wnt ligands are embryonic growth factors that regulate cellular differentiation and survival. We recently demonstrated that canonical and non-canonical Wnt signaling induces RGC survival and axonal regrowth after optic nerve crush (ONC) injury in mouse. Here, we investigated whether the non-canonical Wnt5a ligand induces pro-regenerative inflammation after ONC. Mice were intravitreally injected with Wnt5a or saline during ONC and retina tissue was collected for QPCR and immunofluorescence. We demonstrated that expression of arginase 1, a marker of anti-inflammatory microglia, was upregulated by Wnt5a in injured retinas, whereas iNOS, a marker of neurotoxic microglia, was suppressed. Wnt5a also induced time-dependent changes in pro-inflammatory genes Gal3, TNFα, P2RY12 and IL-6 and the anti-inflammatory gene IL-27. These results indicate that Wnt5a is an immunomodulatory ligand in the retina after ONC injury.

Diabetic retinopathy (DR) is characterized by microvascular damage and increased vascular permeability in the retina. The investigation of visual outcomes in late-stage DR is limited by challenges of maintaining chronically hyperglycemic mice and most reports are restricted to early-stage DR. In this study, we used carefully managed diabetic mice to longitudinally investigate associations between vascular leakage and visual acuity during early and late-stage DR. Diabetes was induced in C57Bl/6J mice with streptozotocin (STZ) and fluorescence angiography with dual fluorescence (FA-DF) was used to assess retinal vascular leakage dynamics in chronically hyperglycemic mice for 12-months. Retinal vascular leakage was evident 180 days after diabetes induction and before reduced visual acuity, measured using the optokinetic response, and vascular leakage continued to increase during DR progression. Mice were also treated with intravitreal injections of anti-angiogenic aflibercept at late-stage DR, and reduced leakage was reliably measured using FA-DF and was associated with improved visual acuity. Inflammatory and vascular phenotypes were assessed using immunostaining, which revealed significantly lower retinal macrophage and vascular densities and reduced capillary diameter in association with anti-VEGF treatment compared to age-matched diabetic controls. In conclusion, this is the first longitudinal quantification of retinal vascular leakage in early, intermediate, and late stages of DR in the same cohort of mice in a minimally invasive fashion, to demonstrate the associated effect of anti-angiogenic therapy in vivo. Our findings also further confirmed the sensitivity of FA-DF in assessing retinal vascular leakage in conjunction with other functional measures in longitudinal studies in the same animals.

Inherited retinal diseases (IRDs) are a large heterogeneous group of diseases that lead to visual impairment and complete vision loss. Retinitis pigmentosa (RP) is an IRD with progressive degeneration of photoreceptors and has been associated with mutations in over 80 genes. In this study, we investigated the mechanism of retinal degeneration caused by an inherited mutation in the Pde6b gene in the rd10 mouse model of RP, with a focus on alternative programmed cell death pathways. RNA-seq analysis was used to identify changes in gene expression in rd10 mice, using C57BL/6J mice as non-degenerating genetic background controls. The functional role of differentially expressed genes was investigated using pharmacological treatments and visual acuity was assessed using optomotor kinetic tracking assay. We found increased expression of genes involved in inflammatory response, while expression of genes involved in photoreceptor function and homeostasis were decreased. We also demonstrated increased expression of genes that regulate oxytosis/ferroptosis, a type of regulated necrosis that can promote inflammatory responses. We found no significant changes in expression of genes controlling other types of regulated necrosis. Treating rd10 mice with oxytosis/ferroptosis inhibitors led to significant improvements in visual acuity. Therefore, these findings suggest that disruption of Pde6b activity results in photoreceptor death via oxytosis/ferroptosis, contributing to inflammatory responses in the retina. Our results identify for the first time a possible role of oxytosis/ferroptosis in a model of inherited retinal degeneration and provide a foundation for further studies exploring oxytosis/ferroptosis inhibitors as a potential therapeutic strategy for RP.

Canonical and non-canonical Wnt signaling pathways are well-characterized regulators of retinal development. Wnt signaling also promotes neuroprotection and regeneration in adult tissues, including retinal ganglion cell (RGC) survival and axonal regrowth after optic nerve injury. However, it is unknown whether Wnt-dependent neuroprotection after injury in the adult CNS is associated with altered expression of developmental genes. Müller glia are a prominent radial glia type in the retina that play critical roles in retinal neuron protection, RGC neurite growth, and axon regeneration by acting through Wnt and other signaling pathways. We recently used mass spectrometry to characterize proteins secreted from Müller glia in response to Wnt signaling. In this study, we investigated whether the Wnt-induced Müller glia secretome includes proteins involved in development and whether their corresponding genes are regulated by Wnt5a during axonal regeneration in a mouse model of optic nerve crush (ONC) injury. Adult mice received intravitreal injections of Wnt5a or saline at the time of ONC injury, and then retina tissue was collected at early time points post-injury. The expression of candidate Wnt-regulated developmental genes and related proteins were characterized by qPCR and immunohistochemistry. Our findings revealed that Wnt5a downregulated the expression of specific developmental genes, including cilia-related genes Nphp4, INTU, and Jade1, as well as transcriptional regulators Pax6 and Tsc1, with time-dependent changes observed during axonal regrowth. Several of these genes were localized to RGCs and inner nuclear layer cells, suggesting direct effects in RGCs and contributions from Müller glia. These results demonstrate that specific developmental gene pathways are suppressed by Wnt5a in association with RGC survival and axon regrowth following injury. Therefore, this study adds to our knowledge of potential mechanisms of Wnt-mediated optic nerve regeneration and identifies new categories of putative regeneration-regulating genes for further study.

In this review, we explore the connections between developmental embryology and axonal regeneration. Genes that regulate embryogenesis and central nervous system (CNS) development are discussed for their therapeutic potential to induce axonal and cellular regeneration in adult tissues after neuronal injury. Despite substantial differences in the tissue environment in the developing CNS compared with the injured CNS, recent studies have identified multiple molecular pathways that promote axonal growth in both scenarios. We describe various molecular cues and signaling pathways involved in neural development, with an emphasis on the versatile Wnt signaling pathway. We discuss the capacity of developmental factors to initiate axonal regrowth in adult neural tissue within the challenging environment of the injured CNS. Our discussion explores the roles of Wnt signaling and also examines the potential of other embryonic genes including Pax, BMP, Ephrin, SOX, CNTF, PTEN, mTOR and STAT3 to contribute to axonal regeneration in various CNS injury model systems, including spinal cord and optic crush injuries in mice, Xenopus and zebrafish. Additionally, we describe potential contributions of Müller glia redifferentiation to neuronal regeneration after injury. Therefore, this review provides a comprehensive summary of the state of the field, and highlights promising research directions for the potential therapeutic applications of specific embryologic molecular pathways in axonal regeneration in adults.

Transcriptomes and proteomes can be normalized with a handful of RNAs or proteins (or their peptides), such as GAPDH, β-actin, RPBMS, and/or GAP43. Even with hundreds of standards, normalization cannot be achieved across different molecular mass ranges for small molecules, such as lipids and metabolites, due to the non-linearity of mass by charge ratio for even the smallest part of the spectrum. We define the amount (or range of amounts) of metabolites and/or lipids per a defined amount of a protein, consistently identified in all samples of a multiple-model organism comparison, as the normative level of that metabolite or lipid. The defined protein amount (or range) is a normalized value for one cohort of complete samples for which intrasample relative protein quantification is available. For example, the amount of citrate (a metabolite) per µg of aconitate hydratase (normalized protein amount) identified in the proteome is the normative level of citrate with aconitase. We define normativity as the amount of metabolites (or amount range) detected when compared to normalized protein levels. We use axon regeneration as an example to illustrate the need for advanced approaches to the normalization of proteins. Comparison across different pharmacologically induced axon regeneration mouse models entails the comparison of axon regeneration, studied at different time points in several models designed using different agents. For the normalization of the proteins across different pharmacologically induced models, we perform peptide doping (fixed amounts of known peptides) in each sample to normalize the proteome across the samples. We develop Regen V peptides, divided into Regen III (SEB, LLO, CFP) and II (HH4B, A1315), for pre- and post-extraction comparisons, performed with the addition of defined, digested peptides (bovine serum albumin tryptic digest) for protein abundance normalization beyond commercial labeled relative quantification (for example, 18-plex tandem mass tags). We also illustrate the concept of normativity by using this normalization technique on regenerative metabolome/lipidome profiles. As normalized protein amounts are different in different biological states (control versus axon regeneration), normative metabolite or lipid amounts are expected to be different for specific biological states. These concepts and standardization approaches are important for the integration of different datasets across different models of axon regeneration.

The optic nerve contains retinal ganglion cell (RGC) axons and functions to transmit visual stimuli to the brain. Injury to the optic nerve from ischemia, trauma, or disease leads to retrograde axonal degeneration and subsequent RGC dysfunction and death, causing irreversible vision loss. Inflammatory responses to neurological damage and axonal injuries in the central nervous system (CNS) are typically harmful to neurons and prevent recovery. However, recent evidence indicates that certain inflammatory cell types and signaling pathways are protective after optic nerve injury and promote RGC survival and axonal regeneration. The objective of this review is to examine the evidence for diverse effects of inflammatory cell types on the retina and optic nerve after injury. Additionally, we highlight promising avenues for further research.

BACKGROUND:Diabetic retinopathy (DR) afflicts more than 93 million people worldwide and is a leading cause of vision loss in working adults. While DR therapies are available, early DR development may go undetected without treatment due to the lack of sufficiently sensitive tools. Therefore, early detection is critically important to enable efficient treatment before progression to vision-threatening complications. A major clinical manifestation of early DR is retinal vascular leakage that may progress from diffuse to more localized focal leakage, leading to increased retinal thickness and diabetic macular edema (DME). In preclinical research, a hallmark of DR in mouse models is diffuse retinal leakage without increased thickness or DME, which limits the utility of optical coherence tomography and fluorescein angiography (FA) for early detection. The Evans blue assay detects diffuse leakage but requires euthanasia, which precludes longitudinal studies in the same animals. METHODS:We developed a new modality of ratiometric fluorescence angiography with dual fluorescence (FA-DF) to reliably detect and longitudinally quantify diffuse retinal vascular leakage in mouse models of induced and spontaneous DR. RESULTS:These studies demonstrated the feasibility and sensitivity of FA-DF in detecting and quantifying retinal vascular leakage in the same mice over time during DR progression in association with chronic hyperglycemia and age. CONCLUSIONS:These proof-of-concept studies demonstrated the promise of FA-DF as a minimally invasive method to quantify DR leakage in preclinical mouse models longitudinally.

Purpose: There is increasing interest in nonpharmacologic approaches to protect retinal ganglion cells (RGCs) after injury and enhance the efficacy of therapeutic molecules. Accumulating evidence demonstrates neuroprotection by the high-fat low-carbohydrate ketogenic diet (KD) in humans and animal models of neurologic diseases. However, no studies to date have examined whether the KD protects RGCs and promotes axonal regrowth after traumatic injury to the optic nerve (ON) or whether it increases efficacy of experimental proregenerative molecules. In this study, we investigated whether the KD promoted RGC survival and axonal regeneration after ON injury in the presence and absence of neuroprotective Wnt3a ligand. Methods: Adult mice were placed on a KD or control diet before ON crush injury and remained on the diet until the end of the experiment. Nutritional ketosis was confirmed by measuring serum beta-hydroxybutyrate levels. Mice were intravitreally injected with Wnt3a ligand or phosphate-buffered saline (PBS), and RGC survival, function, axonal regeneration, and inflammatory responses were measured. Results: Mice fed the KD showed increased RGC survival and reduced inflammatory cells in PBS-injected mice. Also, mice fed the KD had increased RGC functional responses but not increased RGC numbers in the presence of Wnt3a, indicating that the KD did not enhance the prosurvival effect of Wnt3a. The KD did not promote axonal regeneration in the presence or absence of Wnt3a. Conclusions: The KD has a complex protective effect after ON injury and cotreatment with Wnt3a. This work sets the foundation for studies identifying underlying molecular mechanisms.

Canonical and noncanonical Wnt signaling pathways are essential for development and maintenance of the CNS. Whereas the roles of canonical Wnt pathways in neuronal survival and axonal regeneration in adult CNS have been described, the functions of noncanonical Wnt pathways are not well understood. Furthermore, the role of noncanonical Wnt ligands in the adult retina has not been investigated. Noncanonical Wnt signaling shares receptors with canonical Wnt ligands but functions through calcium and c-Jun N-terminal kinase (JNK) signaling pathways. Noncanonical ligands, such as the prototypic ligand Wnt5a, have varying effects in the developing CNS, including inhibiting or promoting axonal growth. To identify a role for noncanonical Wnt signaling in the developed retina after injury, we characterized the effect of Wnt5a on neurite outgrowth in cultured retinal ganglion cell (RGC) neurons and on axonal regeneration in the injured optic nerve in the mouse. Endogenous Wnt5a was upregulated after injury and exogenous Wnt5a significantly enhanced neurite growth of primary RGCs and led to extensive axonal regeneration after optic nerve crush (ONC) injury. Wnt5a also significantly increased RGC survival. Furthermore, Wnt5a induced phosphorylation of CamKII and JNK and induced expression of their downstream pathway components. Therefore, these results demonstrate for the first time that Wnt5a promotes axonal growth and protects RGCs in the adult retina.

Interleukin-27 is a pleiotropic cytokine that is involved in tissue responses to infection, cell stress, neuronal disease, and tumors. Recent studies in various tissues indicate that interleukin-27 has complex activating and inhibitory properties in innate and acquired immunity. The availability of recombinant interleukin-27 protein and mice with genetic deletions of interleukin-27, its receptors and signaling mediators have helped define the role of interleukin-27 in neurodegenerative diseases. Interleukin-27 has been well-characterized as an important regulator of T cell activation and differentiation that enhances or suppresses T cell responses in autoimmune conditions in the central nervous system. Evidence is also accumulating that interleukin-27 has neuroprotective activities in the retina and brain. Interleukin-27 is secreted from and binds to infiltrating microglia, macrophage, astrocytes, and even neurons and it promotes neuronal survival by regulating pro- and anti-inflammatory cytokines, neuroinflammatory pathways, oxidative stress, apoptosis, autophagy, and epigenetic modifications. However, interleukin-27 can have the opposite effect and induce inflammation and cell death in certain situations. In this review, we describe the current understanding of regulatory activities of interleukin-27 on cell survival and inflammation and discuss its mechanisms of action in the brain, spinal cord, and retina. We also review evidence for and against the therapeutic potential of interleukin-27 for dampening harmful neuroinflammatory responses in central nervous system diseases.