Chemokine-mediated inflammation in the degenerating retina is coordinated by Müller cells, activated microglia, and retinal pigment epithelium

BACKGROUND Monocyte infiltration is involved in the pathogenesis of many retinal degenerative conditions. This process traditionally depends on local expression of chemokines, though the roles of many of these in the degenerating retina are unclear. Here, we investigate expression and in situ localization of the broad chemokine response in a light-induced model of retinal degeneration. METHODS Sprague-Dawley (SD) rats were exposed to 1,000 lux light damage (LD) for up to 24 hrs. At time points during (1 to 24 hrs) and following (3 and 7 days) exposure, animals were euthanized and retinas processed. Microarray analysis assessed differential expression of chemokines. Some genes were further investigated using polymerase chain reaction (PCR) and in situ hybridization and contrasted with photoreceptor apoptosis using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Recruitment of retinal CD45 (+) leukocytes was determined via fluorescence activated cell sorting (FACS), and expression of chemokine receptors determined using PCR. RESULTS Exposure to 24 hrs of LD resulted in differential expression of chemokines including Ccl3, Ccl4, Ccl7, Cxcl1, and Cxcl10. Their upregulation correlated strongly with peak photoreceptor death, at 24 hrs exposure. In situ hybridization revealed that the modulated chemokines were expressed by a combination of Müller cells, activated microglia, and retinal pigment epithelium (RPE). This preceded large increases in the number of CD45(+) cells at 3- and 7-days post exposure, which expressed a corresponding repertoire of chemokine receptors. CONCLUSIONS Our data indicate that retinal degeneration induces upregulation of a broad chemokine response whose expression is coordinated by Müller cells, microglia, and RPE. The findings inform our understanding of the processes govern the trafficking of leukocytes, which are contributors in the pathology of retinal degenerations

Anti-inflammatory and neuroprotective properties of the corticosteroid fludrocortisone in retinal degeneration

The pathogenesis of outer retinal degenerations has been linked to the elevation of cytokines that orchestrate pro-inflammatory responses within the retinal milieu, and which are thought to play a role in diseases such as geographic atrophy (GA), an advanced form of AMD. Here we sought investigate the anti-inflammatory and mechanistic properties of fludrocortisone (FA), as well as triamcinolone acetonide (TA), on Müller cell-mediated cytokine expression in response to inflammatory challenge. In addition, we investigated the neuroprotective efficacy of FA and TA in a photo-oxidative damage (PD), a model of outer retinal degeneration. Expression of CCL2, IL-6, and IL-8 with respect to FA and TA were assessed in Müller cells in vitro, following simulation with IL-1β or TNF-α. The dependency of this effect on mineralocorticoid and glucocorticoid signaling was also interrogated for both TA and TA via co-incubation with steroid receptor antagonists. For the PD model, C57BL/6 mice were intravitreally injected with FA or TA, and changes in retinal pathology were assessed via electroretinogram (ERG) and optical coherence tomography (OCT). FA and TA were found to dramatically reduce the expression of CCL2, IL-6, and IL-8 in Müller glia in vitro after inflammatory challenge with IL-1β or TNF-α (P  0.05). Our data indicate potent anti-inflammatory and mechanistic properties of corticosteroids, specifically FA, in suppressing inflammation and neurodegeneration degeneration associated with outer retinal atrophy. Taken together, our findings indicate that corticosteroids such as FA may have value as a potential therapeutic for outer retinal degenerations where such pro-inflammatory factors are implicated, including AMD

Synthesis and propagation of complement C3 by microglia/monocytes in the aging retina

INTRODUCTION Complement activation is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which may be mediated in part by para-inflammatory processes. We aimed to investigate the expression and localization of C3, a crucial component of the complement system, in the retina during the course of aging. METHODS SD rats were born and reared in low-light conditions, and euthanized at post-natal (P) days 100, 450, or 750. Expression of C3, IBA1, and Ccl- and Cxcl- chemokines was assessed by qPCR, and in situ hybridization. Thickness of the ONL was assessed in retinal sections as a measure of photoreceptor loss, and counts were made of C3-expressing monocytes. RESULTS C3 expression increased significantly at P750, and correlated with thinning of the ONL, at P750, and up-regulation of GFAP. In situ hybridization showed that C3 was expressed by microglia/monocytes, mainly from within the retinal vasculature, and occasionally the ONL. The number of C3-expressing microglia increased significantly by P750, and coincided spatiotemporally with thinning of the ONL, and up-regulation of Ccl- and Cxcl- chemokines. CONCLUSIONS Our data suggest that recruited microglia/monocytes contribute to activation of complement in the aging retina, through local expression of C3 mRNA. C3 expression coincides with age-related thinning of the ONL at P750, although it is unclear whether the C3-expressing monocytes are a cause or consequence. These findings provide evidence of activation of complement during natural aging, and may have relevance to cellular events underling the pathogenesis of age-related retinal diseases.Funding provided by Australian Research Council Centres of Excellence Program Grant (CE0561903)

Analysis of complement expression in light-induced retinal degeneration: Synthesis and deposition of C3 by microglia/macrophages is associated with focal photoreceptor degeneration

Purpose. To investigate the expression and localization of complement system mRNA and protein in a light-induced model of progressive retinal degeneration. Methods. Sprague-Dawley (SD) rats were exposed to 1000 lux of bright continuous light (BCL) for up to 24 hours. At time points during (1-24 hours) and after (3 and 7 days) exposure, the animals were euthanatized and the retinas processed. Differential expression of complement genes at 24 hours of exposure was assessed using microarray analysis. Expression of complement genes was validated by quantitative PCR, and expression of selected genes was investigated during and after BCL exposure. Photoreceptor apoptosis was assessed using TUNEL and C3 was further investigated by spatiotemporal analysis using in situ hybridization and immunohistochemistry. Results. Exposure to 24 hours of BCL induced differential expression of a suite of complement system genes, including classic and lectin components, regulators, and receptors. C1qr1, MCP, Daf1, and C1qTNF6 all modulated in concert with photoreceptor death and AP-1 expression, which reached a peak at 24 hours exposure. C1s and C4a reached peak expression at 3 days after exposure, while expression of C3, C3ar1, and C5r1 were maximum at 7 days after exposure. C3 mRNA was detected in ED1- and IBA1-positive microglia/macrophages, in the retinal vessels and optic nerve head and in the subretinal space, particularly at the margins of the emerging lesion. Conclusions. The data indicate that BCL induces the prolonged expression of a range of complement genes and show that microglia/macrophages synthesize C3 and deposit it in the ONL after BCL injury. These findings have relevance to the role of complement in progressive retinal degeneration, including atrophic AMD

670-nm light treatment reduces complement propagation following retinal degeneration

AIM: Complement activation is associated with the pathogenesis of age-related macular degeneration (AMD). We aimed to investigate whether 670-nm light treatment reduces the propagation of complement in a light-induced model of atrophic AMD. METHODS: Sprague–Dawley (SD) rats were pretreated with 9 J/cm(2) 670-nm light for 3 minutes daily over 5 days; other animals were sham treated. Animals were exposed to white light (1,000 lux) for 24 h, after which animals were kept in dim light (5 lux) for 7 days. Expression of complement genes was assessed by quantitative polymerase chain reaction (qPCR), and immunohistochemistry. Counts were made of C3-expressing monocytes/microglia using in situ hybridization. Photoreceptor death was also assessed using outer nuclear layer (ONL) thickness measurements, and oxidative stress using immunohistochemistry for 4-hydroxynonenal (4-HNE). RESULTS: Following light damage, retinas pretreated with 670-nm light had reduced immunoreactivity for the oxidative damage maker 4-HNE in the ONL and outer segments, compared to controls. In conjunction, there was significant reduction in retinal expression of complement genes C1s, C2, C3, C4b, C3aR1, and C5r1 following 670 nm treatment. In situ hybridization, coupled with immunoreactivity for the marker ionized calcium binding adaptor molecule 1 (IBA1), revealed that C3 is expressed by infiltrating microglia/monocytes in subretinal space following light damage, which were significantly reduced in number after 670 nm treatment. Additionally, immunohistochemistry for C3 revealed a decrease in C3 deposition in the ONL following 670 nm treatment. CONCLUSIONS: Our data indicate that 670-nm light pretreatment reduces lipid peroxidation and complement propagation in the degenerating retina. These findings have relevance to the cellular events of complement activation underling the pathogenesis of AMD, and highlight the potential of 670-nm light as a non-invasive anti-inflammatory therapy

Measurement of the radiative decay of polarized muons in the MEG experiment

We studied the radiative muon decay $\mu^+ \to e^+\nu\bar{\nu}\gamma$ by using for the first time an almost fully polarized muon source. We identified a large sample (~13000) of these decays in a total sample of 1.8x10^14 positive muon decays collected in the MEG experiment in the years 2009--2010 and measured the branching ratio B($\mu^+ \to e^+\nu\bar{\nu}\gamma$) = (6.03+-0.14(stat.)+-0.53(sys.))x10^-8 for E_e > 45 MeV and E_{\gamma} > 40 MeV, consistent with the Standard Model prediction. The precise measurement of this decay mode provides a basic tool for the timing calibration, a normalization channel, and a strong quality check of the complete MEG experiment in the search for $\mu^+ \to e^+\gamma$ process.Comment: 8 pages, 7 figures. Added an introduction to NLO calculation which was recently calculated. Published versio

Molecular nitrogen in N doped TiO2 nanoribbons

The nitrogen doping of TiO2 nanoribbons during the thermal transformation of hydrogen titanate nanoribbons HTiNRs between 400 and 650 C in a dynamic ammonia atmosphere was investigated using X ray photoelectron spectroscopy XPS , transmission X ray microscopy combined with near edge X ray absorption fine structure spectroscopy NEXAFS TXM , X ray diffraction XRD and electron paramagnetic resonance measurements EPR . Comprehensive structural characterizations have revealed that for a calcination temperature of 400 C, the HTiNRs transform into pure monoclinic TiO2 b phase TiO2 B whereas at higher calcination temperatures 580 and 650 C a mixture of TiO2 B and anatase is obtained. XPS and EPR results clearly reveal the nitrogen doping of TiO2 nanoribbons and that, depending on the calcination temperature, nitrogen atoms occupy interstitial and substitutional sites. Moreover, in samples calcined at 580 and 650 C the presence of N2 like species in the HTiNRs was detected by NEXAFS TXM. These species are trapped in the HTiNRs structure. EPR measurements upon light illumination have disclosed the generation of photoexcited states which implies that nitrogen has an important effect on the electronic structure of N doped TiO

Initial experience with an electron FLASH research extension (FLEX) for the Clinac system

Purpose: Radiotherapy delivered at ultra-high-dose-rates (≥40 Gy/s), that is, FLASH, has the potential to effectively widen the therapeutic window and considerably improve the care of cancer patients. The underlying mechanism of the FLASH effect is not well understood, and commercial systems capable of delivering such dose rates are scarce. The purpose of this study was to perform the initial acceptance and commissioning tests of an electron FLASH research product for preclinical studies. Methods: A linear accelerator (Clinac 23EX) was modified to include a nonclinical FLASH research extension (the Clinac-FLEX system) by Varian, a Siemens Healthineers company (Palo Alto, CA) capable of delivering a 16 MeV electron beam with FLASH and conventional dose rates. The acceptance, commissioning, and dosimetric characterization of the FLEX system was performed using radiochromic film, optically stimulated luminescent dosimeters, and a plane-parallel ionization chamber. A radiation survey was conducted for which the shielding of the pre-existing vault was deemed sufficient. Results: The Clinac-FLEX system is capable of delivering a 16 MeV electron FLASH beam of approximately 1 Gy/pulse at isocenter and reached amaximum dose rate \u3e3.8 Gy/pulse near the upper accessory mount on the linac gantry. The percent depth dose curves of the 16 MeV FLASH and conventional modes for the 10 × 10 cm2 applicator agreed within 0.5 mm at a range of 50% of the maximum dose. Their respective profiles agreed well in terms of flatness but deviated for field sizes \u3e10 × 10 cm2. The output stability of the FLASH system exhibited a dose deviation of \u3c1%.Preliminary cell studies showed that the FLASH dose rate (180 Gy/s) had much less impact on the cell morphology of 76N breast normal cells compared to the non-FLASH dose rate (18 Gy/s), which induced large-size cells. Conclusion: Our studies characterized the non-clinical Clinac-FLEX system as a viable solution to conduct FLASH research that could substantially increase access to ultra-high-dose-rate capabilities for scientists

Three-year survival of single- and two-surface ART restorations in a high-caries child population

The aim of this study was to evaluate the survival of single- and two-surface atraumatic restorative treatment (ART) restorations in the primary and permanent dentitions of children from a high-caries population, in a field setting. The study was conducted in the rainforest of Suriname, South America. ART restorations, made by four Dutch dentists, were evaluated after 6 months, 1, 2, and 3 years. Four hundred seventy-five ART restorations were placed in the primary dentition and 54 in first permanent molars of 194 children (mean age 6.09 ± 0.48 years). Three-year cumulative survivals of single- and two-surface ART restorations in the primary dentition were 43.4 and 12.2%, respectively. Main failure characteristics were gross marginal defects and total or partial losses. Three-year cumulative survival for single-surface ART restorations in the permanent dentition was 29.6%. Main failure characteristics were secondary caries and gross marginal defects. An operator effect was found only for two-surface restorations. The results show extremely low survival rates for single- and two-surface ART restorations in the primary and permanent dentitions. The variable success for ART may initiate further discussion about alternative treatment strategies, especially in those situations where choices have to be made with respect to a well-balanced, cost-effective package of basic oral health care