Concept of Normativity in Multi-Omics Analysis of Axon Regeneration.

Transcriptomes and proteomes can be normalized with a handful of RNAs or proteins (or their peptides), such as GAPDH, ß-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.

Repurposing development genes for axonal regeneration following injury: Examining the roles of Wnt signaling.

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.

Context-Dependent Effects of the Ketogenic Diet on Retinal Ganglion Cell Survival and Axonal Regeneration After Optic Nerve Injury.

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.

Fluorescence Angiography with Dual Fluorescence for the Early Detection and Longitudinal Quantitation of Vascular Leakage in Retinopathy.

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.

Assessment of outer retinal thickness and function in mice after experimental optic nerve trauma.

BACKGROUND: Optic nerve trauma caused by crush injury is frequently used for investigating experimental treatments that protect retinal ganglion cells (RGCs) and induce axonal regrowth. Retaining outer retinal light responses is essential for therapeutic rescue of RGCs after injury. However, whether optic nerve crush also damages the structure or function of photoreceptors has not been systematically investigated. In this study, we investigated whether outer retinal thickness and visual function are altered by optic nerve crush in the mouse. METHODS: Wildtype mice underwent optic nerve crush and intravitreal injection of a control solution in one eye with the fellow eye remaining uninjured. Two weeks after injury, the thickness of the ganglion cell region (GCL to IPL) and photoreceptor layer (bottom of the OPL to top of the RPE) were measured using OCT. Retinal function was assessed using flash ERGs. Immunodetection of RGCs was performed on retinal cryosections and RGCs and ONL nuclei rows were counted. Multiple comparison analyses were conducted using Analysis of Variance (ANOVA) with Tukey's post hoc test and P values less than 0.05 were considered statistically significant. RESULTS: Optic nerve crush injury induced RGC death as expected, demonstrated by thinning of the ganglion cell region and RGC loss. In contrast, outer retinal thickness, photopic and scotopic a-wave and b-wave amplitudes and photoreceptor nuclei counts, were equivalent between injured and uninjured eyes. CONCLUSIONS: Secondary degeneration of the outer retina was not detected after optic nerve injury in the presence of significant RGC death, suggesting that the retina has the capacity to compartmentalize damage. These findings also indicate that experimental treatments to preserve the GCL and rescue vision using this optic nerve injury model would not require additional strategies to preserve the ONL.

The cytokine IL-27 reduces inflammation and protects photoreceptors in a mouse model of retinal degeneration.

BACKGROUND: Retinal degenerative diseases are a group of conditions characterized by photoreceptor death and vision loss. Excessive inflammation and microglial activation contribute to the pathology of retinal degenerations and a major focus in the field is identifying more effective anti-inflammatory therapeutic strategies that promote photoreceptor survival. A major challenge to developing anti-inflammatory treatments is to selectively suppress detrimental inflammation while maintaining beneficial inflammatory responses. We recently demonstrated that endogenous levels of the IL-27 cytokine were upregulated in association with an experimental treatment that increased photoreceptor survival. IL-27 is a pleiotropic cytokine that regulates tissue reactions to infection, neuronal disease and tumors by inducing anti-apoptotic and anti-inflammatory genes and suppressing pro-inflammatory genes. IL-27 is neuroprotective in the brain, but its function during retinal degeneration has not been investigated. In this study, we investigated the effect of IL-27 in the rd10 mouse model of inherited photoreceptor degeneration. METHODS: Male and female rd10 mice were randomly divided into experimental (IL-27) and control (saline) groups and intravitreally injected at age post-natal day (P) 18. Retina function was analyzed by electroretinograms (ERGs), visual acuity by optomotor assay, photoreceptor death by TdT-mediated dUTP nick-end labeling (TUNEL) assay, microglia/macrophage were detected by immunodetection of IBA1 and inflammatory mediators by cytoplex and QPCR analysis. The distribution of IL-27 in the retina was determined by immunohistochemistry on retina cross-sections and primary Muller glia cultures. RESULTS: We demonstrate that recombinant IL-27 decreased photoreceptor death, increased retinal function and reduced inflammation in the rd10 mouse model of retinal degeneration. Furthermore, IL-27 injections led to lower levels of the pro-inflammatory proteins Ccl22, IL-18 and IL-12. IL-27 expression was localized to Muller glia and IL-27 receptors to microglia, which are key cell types that regulate photoreceptor survival. CONCLUSION: Our results identify for the first time anti-inflammatory and neuroprotective activities of IL-27 in a genetic model of retinal degeneration. These findings provide new insight into the therapeutic potential of anti-inflammatory cytokines as a treatment for degenerative diseases of the retina.

Identification of a Novel Axon Regeneration Role for Noncanonical Wnt Signaling in the Adult Retina after Injury.

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.

Exploring the role of interleukin-27 as a regulator of neuronal survival in central nervous system diseases.

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.

Neuroprotection by the Ketogenic Diet: Evidence and Controversies.

The ketogenic diet (KD) is a high-fat low-carbohydrate diet that has been used for decades as a non-pharmacologic approach to treat metabolic disorders and refractory pediatric epilepsy. In recent years, enthusiasm for the KD has increased in the scientific community due to evidence that the diet reduces pathology and improves various outcome measures in animal models of neurodegenerative disorders, including multiple sclerosis, stroke, glaucoma, spinal cord injury, retinal degenerations, Parkinson's disease and Alzheimer's disease. Clinical trials also suggest that the KD improved quality of life in patients with multiple sclerosis and Alzheimer's disease. Furthermore, the major ketone bodies BHB and ACA have potential neuroprotective properties and are now known to have direct effects on specific inflammatory proteins, transcription factors, reactive oxygen species, mitochondria, epigenetic modifications and the composition of the gut microbiome. Neuroprotective benefits of the KD are likely due to a combination of these cellular processes and other potential mechanisms that are yet to be confirmed experimentally. This review provides a comprehensive summary of current evidence for the effectiveness of the KD in humans and preclinical models of various neurological disorders, describes molecular mechanisms that may contribute to its beneficial effects, and highlights key controversies and current gaps in knowledge.

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.

Association of the Indoor Environment With Dry Eye Metrics.

Importance: The ocular surface is continuously exposed to the environment. Although studies have focused on associations between outdoor environmental conditions and dry eye, information on associations between the indoor environment and dry eye is lacking. Objective: To determine associations between the indoor environment and dry eye. Design, Setting, and Participants: This prospective cross-sectional study sample of 97 veterans with a wide range of dry eye metrics was recruited from the Miami Veterans Affairs Healthcare eye clinic from October 19, 2017, to August 30, 2018. Dry eye metrics were first evaluated in the clinic, followed by indoor home environmental metrics within 1 week using a handheld particle counter. Data were analyzed from October 19, 2017, to August 30, 2018. Main Outcomes and Measures: Symptoms of dry eye were assessed with standardized questionnaires. Dry eye signs were assessed via standard examination. Indoor environmental metrics included temperature, humidity, and particulate matter mass and count. Results: Of the 97 participants included in the analysis, 81 (84%) were men, with a mean (SD) age of 58.2 (11.9) years. Dry eye symptoms were in the moderate range with a mean (SD) Ocular Surface Disease Index (OSDI) score of 31.2 (23.6). Humidity was associated with worse symptoms and signs, including OSDI score (r = 0.30 [95% CI, 0.07-0.49]; P = .01), inflammation (r = 0.32 [95% CI, 0.10-0.51]; P = .01), Schirmer score (r = -0.25 [95% CI, -0.45 to 0.02]; P = .03), eyelid vascularity (r = 0.27 [95% CI, 0.05-0.47]; P = .02), and meibomian gland dropout (r = 0.27 [95% CI, 0.05-0.47]; P = .02). In multivariate analyses, particulate matter of 2.5 µm or less (PM2.5) was associated with dry eye metrics when adjusted for demographic characteristics, comorbidities, medications, and interaction variables. For example, a 1-unit increase in instrumented PM2.5 level was associated with a 1.59 increase in the OSDI score (95% CI, 0.58-2.59; P = .002), a 0.39 reduction in Schirmer score (95% CI, -0.75 to -0.03; P = .04), a 0.07 increase in meibomian gland dropout (95% CI, 0.01-0.13; P = .02), and a 0.06 increase in inflammation (95% CI, 0.02-0.11; P = .009). Conclusions and Relevance: When adjusting for humidity, this study found that increased particulate matter exposure was associated with worse dry eye metrics. Humidity was positively associated with dry eye metrics, potentially because higher humidity increases microbial growth and particulate matter size and mass.

The effect of extrinsic Wnt/ß-catenin signaling in Muller glia on retinal ganglion cell neurite growth.

Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/ß-catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co-cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt-dependent target proteins that may mediate the axonal growth.

Increased Neuroprotective Microglia and Photoreceptor Survival in the Retina from a Peptide Inhibitor of Myeloid Differentiation Factor 88 (MyD88).

Myeloid differentiation factor 88 (MyD88) is an adaptor protein for the Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) families of innate immunity receptors that mediate inflammatory responses to cellular injury. TLR/IL1R/MyD88 signaling is known to contribute to retinal degeneration, although how MyD88 regulates neuronal survival, and the effect of MyD88 on the inflammatory environment in the retina, is mostly unknown. In this study, we tested the hypothesis that blocking MyD88-mediated signaling early in retinal degeneration promotes transition of microglia towards a neuroprotective anti-inflammatory phenotype, resulting in enhanced photoreceptor survival. We also tested whether systemic delivery of a pharmacologic MyD88 inhibitor has therapeutic potential. The rd10 mouse model of retinal degeneration was injected intraperitoneally with increasing doses of a MyD88 blocking peptide or control peptide early in degeneration, and inflammatory responses and photoreceptor survival were measured at specific time points using flow cytometry, cytokine profiling, and electroretinograms. Our results demonstrated that rd10 mice injected with a low dose of MyD88 inhibitor peptide showed increased rod photoreceptor function and reduced apoptosis compared with control peptide and uninjected mice. MyD88 inhibition also resulted in fewer microglia/macrophage cells in the photoreceptor layer whereas total peripheral and retinal macrophage were not changed. Furthermore, increased number of cells expressing the Arg1 marker of neuroprotective microglia in the photoreceptor layer and higher MCP-1 and anti-inflammatory cytokine IL-27 were associated with photoreceptor survival. Therefore, these data suggest that the MyD88 inhibitor modified the retina environment to become less inflammatory, leading to improved photoreceptor function and survival.

Multi-Omic Analyses of Growth Cones at Different Developmental Stages Provides Insight into Pathways in Adult Neuroregeneration.

Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which necessitates protein-lipid interactions, is critical to axonal elongation in development and in adult neuritogenesis. We present a multi-omic analysis that integrates proteomics and lipidomics data for the identification of GC pathways, cell phenotypes, and lipid-protein interactions, with an analytic platform to facilitate the visualization of these data. We combine lipidomic data from GC and adult axonal regeneration following optic nerve crush. Our results reveal significant molecular variability in GCs across developmental ages that aligns with the upregulation and downregulation of lipid metabolic processes and correlates with distinct changes in the lipid composition of GC plasmalemma. We find that these processes also define the transition into a growth-permissive state in the adult central nervous system. The insight derived from these analyses will aid in promoting adult regeneration and functional innervation in devastating neurodegenerative diseases.

Total Tear IgE Levels Correlate with Allergenic and Irritating Environmental Exposures in Individuals with Dry Eye.

Dry eye (DE) and allergic conjunctivitis may present similarly, and it remains unclear whether some individuals have an underlying allergic component to their DE. To better understand this relationship, we performed a cross-sectional study in 75 individuals with DE symptoms and/or signs. Immunoglobulin E (IgE) levels in tear samples were quantified and home environmental exposures assessed via standardized survey. Tears were collected by Schirmer strip, and total tear IgE levels were quantified using enzyme-linked immunosorbent assay (ELISA). Data were analyzed using descriptive statistics and linear and logistic regressions. The main outcome measures were total tear IgE levels and their association with environmental exposures. The mean age of the subjects was 66.2 ± 7.8 years. Sixty-two individuals had dry eye symptoms (Dry Eye Questionnaire-5 ≥ 6), and 75 had one or more signs of DE. Detectable total tear IgE levels were observed in 76% of subjects, and 17.3% had high levels (>1 ng/mL). Individuals with exposure to pet(s) (odds ratio (OR) 11.5, p = 0.002) and smoke (OR 38.6, p = 0.008) at home were more likely to have high IgE levels compared to those not exposed. Individuals with tears collected during spring or summer were 3.9 times (p = 0.028) more likely to have high IgE compared to those sampled at other times of year. Subjects born in the US were 3.45 times (p = 0.010) more likely to have high IgE compared to individuals born outside the US. To conclude, a majority of individuals with DE symptoms and/or signs had detectable IgE levels in their tears. High tear IgE levels were correlated with allergy season and exposures in the home linked with allergy.

Lipid profiling dataset of the Wnt3a-induced optic nerve regeneration.

We present lipid profiling data from mouse retina and optic nerve after optic nerve crush and during Wnt3a-induced axonal regeneration at 7 and 15 days post-crush. This data is available at the Metabolomics Workbench, http://www.metabolomicsworkbench.org (Project ID: PR000718).

Human Ocular Surface Particulate Composition in the Clinical Versus Home Environment.

PURPOSE: Our eyes are chronically exposed to airborne particulate matter shown to adversely affect the ocular surface. This research examines size, type (organic vs. inorganic), and elemental composition of particles recovered from the ocular surface in 2 environments and their associations with dry eye (DE) metrics. METHODS: Particles were recovered from the right eye using Schirmer strips obtained both in the clinic and home environments 9 ± 8 days apart. Particle size and elemental composition were assessed using scanning electron microscopy and energy dispersive spectroscopy. The paired t test was used to evaluate the differences in the size and types of ocular surface particles recovered from the clinic and home settings. Associations of particle size and type with home environmental conditions and DE measures were evaluated using correlation analyses. RESULTS: The mean age of the 15 patients was 56 years, standard deviation (±) 12 years; 93% were men and 53% self-identified as white. Size, type, and elemental composition did not vary significantly between clinic and home. Particle surface area was marginally associated with home indoor temperature (25 °C ± 2, ρ=-0.53, P = 0.06) and significantly associated with the select DE signs: tear osmolality (304 mOsm/L ± 14, ρ= -0.60, P = 0.02), inflammation (0.7 ± 0.8, ρ = 0.53, P = 0.04), and tear breakup time (7 seconds ± 3, ρ = 0.56, P = 0.03). CONCLUSIONS: Ocular surface particles were consistently detected across 2 different environments. Greater particle area detected on Schirmer strips correlated with some DE measures, suggesting that particles detected on the ocular surface may affect eye health.

Wnt signaling induces neurite outgrowth in mouse retinal ganglion cells.

Wingless-type (Wnt) signaling pathways mediate axonal growth and remodeling in the embryonic optic nerve, brain and spinal cord. Recent studies demonstrated that the canonical Wnt/ß-catenin signaling pathway also induces axonal regeneration after injury in the optic nerve of adult animals. However, the molecular mechanisms of Wnt-mediated axonal growth are not well understood. Additionally, because Wnt signaling is stimulated in neurons as well as neighboring non-neuronal cells, the cell type(s) responsible for Wnt-induced axonal regeneration are not known. The objectives of this study were to investigate potential mechanisms and target cells of Wnt3a stimulated neurite growth using primary retinal ganglion cell (RGC) cultures. We demonstrated that Wnt3a ligand induced dose-dependent increases in average neurite length and number of neurites in RGCs. QPCR analysis of candidate mediators showed that Wnt3a-dependent neurite growth was associated with lower expression of Ripk1 and Ripk3 genes. Additionally, inhibiting Ripk1 signaling with Necrostatin-1s led to increased neurite number per cell but not increased neurite length. Therefore, Ripk signaling may be involved in mediating the effects of Wnt3a on neurite number but Ripk activity does not seem to be required for Wnt3a-dependent regulation of neurite length. This study shows that RGCs are direct cellular targets of Wnt3a-induced axonal growth, and we identified a novel association between Wnt signaling and Rip kinases in neurite formation.

Impact of seasonal variation in meteorological conditions on dry eye severity.

PURPOSE: To compare dry eye (DE) diagnosis patterns by season in Miami vis-a-vis the US and examine differences in DE symptoms and signs by season in Miami. PATIENTS AND METHODS: US veteran affairs (VA) patient visits with ICD-9 codes for DE (375.15) and routine medical examination (V70.0) from 2010 to 2013 were retrospectively analyzed to evaluate the seasonal pattern of DE diagnosis. A total of 365 patients with normal ocular anatomy were prospectively recruited from the Miami VA eye clinic from 2014 to 2016 for the assessment of symptoms and signs. RESULTS: While DE visit prevalence in Miami was about 10% lower than that of the rest of the country (22.5% vs 33.7%), Miami had roughly four times higher variability in DE visit prevalence throughout the year than the US. Peak values for DE symptoms in the Miami cohort aligned with peak DE prevalence seen in the retrospective sample, occurring in spring and fall. A similar, but less dramatic, pattern was noted with DE signs. The seasonal pattern in DE symptoms remained even after controlling for confounders including demographics and medication use. CONCLUSION: DE symptoms, and to a lesser degree signs, varied by month, with the highest severity of symptoms occurring in spring and fall, which corresponded with peak allergy season and weather fluctuations, respectively. These findings have important implications for season-specific diagnosis, treatment, and management of DE.

A growing field: The regulation of axonal regeneration by Wnt signaling.

The canonical Wnt/ß-catenin pathway is a highly conserved signaling cascade that plays critical roles during embryogenesis. Wnt ligands regulate axonal extension, growth cone guidance and synaptogenesis throughout the developing central nervous system (CNS). Recently, studies in mammalian and fish model systems have demonstrated that Wnt/ß-catenin signaling also promotes axonal regeneration in the adult optic nerve and spinal cord after injury, raising the possibility that Wnt could be developed as a therapeutic strategy. In this review, we summarize experimental evidence that reveals novel roles for Wnt signaling in the injured CNS, and discuss possible mechanisms by which Wnt ligands could overcome molecular barriers inhibiting axonal growth to promote regeneration. A central challenge in the neuroscience field is developing therapeutic strategies that induce robust axonal regeneration. Although adult axons have the capacity to respond to axonal guidance molecules after injury, there are several major obstacles for axonal growth, including extensive neuronal death, glial scars at the injury site, and lack of axonal guidance signals. Research in rodents demonstrated that activation of Wnt/ß-catenin signaling in retinal neurons and radial glia induced neuronal survival and axonal growth, but that activation within reactive glia at the injury site promoted proliferation and glial scar formation. Studies in zebrafish spinal cord injury models confirm an axonal regenerative role for Wnt/ß-catenin signaling and identified the cell types responsible. Additionally, in vitro and in vivo studies demonstrated that Wnt induces axonal and neurite growth through transcription-dependent effects of its central mediator ß-catenin, potentially by inducing regeneration-promoting genes. Canonical Wnt signaling may also function through transcription-independent interactions of ß-catenin with cytoskeletal elements, which could stabilize growing axons and control growth cone movement. Therefore, these studies suggest that Wnt-induced pathways responsible for regulating axonal growth during embryogenesis could be repurposed to promote axonal growth after injury.