Targeting translocator protein (18 kDa) (TSPO) dampens pro-inflammatory microglia reactivity in the retina and protects from degeneration

BACKGROUND: Reactive microglia are commonly seen in retinal degenerative diseases, and neurotoxic microglia responses can contribute to photoreceptor cell death. We and others have previously shown that translocator protein (18 kDa) (TSPO) is highly induced in retinal degenerations and that the selective TSPO ligand XBD173 (AC-5216, emapunil) exerts strong anti-inflammatory effects on microglia in vitro and ex vivo. Here, we investigated whether targeting TSPO with XBD173 has immuno-modulatory and neuroprotective functions in two mouse models of acute retinal degeneration using bright white light exposure.

METHODS: BALB/cJ and Cx3cr1 (GFP/+) mice received intraperitoneal injections of 10 mg/kg XBD173 or vehicle for five consecutive days, starting 1 day prior to exposure to either 15,000 lux white light for 1 h or 50,000 lux focal light for 10 min, respectively. The effects of XBD173 treatment on microglia and Müller cell reactivity were analyzed by immuno-stainings of retinal sections and flat mounts, fluorescence-activated cell sorting (FACS) analysis, and mRNA expression of microglia markers using quantitative real-time PCR (qRT-PCR). Optical coherence tomography (OCT), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stainings, and morphometric analyses were used to quantify the extent of retinal degeneration and photoreceptor apoptosis.

RESULTS: Four days after the mice were challenged with bright white light, a large number of amoeboid-shaped alerted microglia appeared in the degenerating outer retina, which was nearly completely prevented by treatment with XBD173. This treatment also down-regulated the expression of TSPO protein in microglia but did not change the TSPO levels in the retinal pigment epithelium (RPE). RT-PCR analysis showed that the microglia/macrophage markers Cd68 and activated microglia/macrophage whey acidic protein (Amwap) as well as the pro-inflammatory genes Ccl2 and Il6 were reduced after XBD173 treatment. Light-induced degeneration of the outer retina was nearly fully blocked by XBD173 treatment. We further confirmed these findings in an independent mouse model of focal light damage. Retinas of animals receiving XBD173 therapy displayed significantly more ramified non-reactive microglia and more viable arrestin-positive cone photoreceptors than vehicle controls.

CONCLUSIONS: Targeting TSPO with XBD173 effectively counter-regulates microgliosis and ameliorates light-induced retinal damage, highlighting a new pharmacological concept for the treatment of retinal degenerations.

Validation of a high-throughput immunobead array technique for multiplex detection of three foodborne pathogens in chicken products

This study rigorously evaluated a previously developed immunobead array method to simultaneously detect three important foodborne pathogens, Campylobacter jejuni, Listeria monocytogenes, and Salmonella spp., for its actual application in routine food testing. Due to the limitation of the detection limit of the developed method, an enrichment step was included in this study by using Campylobacter Enrichment Broth for C. jejuni and Universal Pre-enrichment Broth for L. monocytogenes and Salmonella spp.. The findings show that the immunobead array method was capable of detecting as low as 1 CFU of the pathogens spiked in the culture media after being cultured for 24 hours for all three pathogens. The immunobead array method was further evaluated for its pathogen detection capabilities in ready-to-eat (RTE) and ready-to-cook (RTC) chicken samples and proven to be able to detect as low as 1 CFU of the pathogens spiked in the food samples after being cultured for 24 hours in the case of Salmonella spp., and L. monocytogenes and 48 hours in the case of C. jejuni. The method was subsequently validated with three types of chicken products (RTE, n=30; RTC, n=20; raw chicken, n=20) and was found to give the same results as the conventional plating method. Our findings demonstrated that the previously developed immunobead array method could be used for actual food testing with minimal enrichment period of only 52 hours, whereas the conventional ISO protocols for the same pathogens take 90-144 hours. The immunobead array was therefore an inexpensive, rapid and simple method for the food testing.

Diabetes Impairs the Aldehyde Detoxifying Capacity of the Retina

Purpose: We studied whether the accumulation of advanced lipoxidation end-products (ALEs) in the diabetic retina is linked to the impairment of lipid aldehyde detoxification mechanisms.

Methods: Retinas were collected from nondiabetic and diabetic rats and processed for conventional and quantitative RT-PCR (qRT-PCR), Western blotting, immunohistochemistry, and aldehyde dehydrogenase (ALDH) activity assays. The effect of the ALDH1a1 inhibitor, NCT-501, on ALE accumulation and cell viability in cultured Müller glia also was investigated.

Results: The rat retina expressed a range of lipid aldehyde detoxifying ALDH and aldo-keto reductase (AKR) genes. In diabetes, mRNA levels were reduced for 5 of 9 transcripts tested. These findings contrasted with those in the lens and cornea where many of these enzymes were upregulated. We have reported previously accumulation of the acrolein (ACR)-derived ALE, FDP-lysine, in retinal Müller glia during diabetes. In the present study, we show that the main ACR-detoxifying ALDH and AKR genes expressed in the retina, namely, ALDH1a1, ALDH2, and AKR1b1, are principally localized to Müller glia. Diabetes-induced FDP-lysine accumulation in Müller glia was associated with a reduction in ALDH1a1 mRNA and protein expression in whole retina and a decrease in ALDH1a1-immunoreactivity specifically within these cells. No such changes were detected for ALDH2 or AKR1b1. Activity of ALDH was suppressed in the diabetic retina and blockade of ALDH1a1 in cultured Müller glia triggered FDP-lysine accumulation and reduced cell viability.

Conclusions: These findings suggest that downregulation of ALDH and AKR enzymes, particularly ALDH1a1, may contribute ALE accumulation in the diabetic retina.

Retinal Pigment Epithelial Cell DNA is Damaged by Exposure to Benzo[a]pyrene, a Constituent of Cigarette Smoke.

This study examined the effect of exogenous benzo[ a ]pyrene (BaP), an important constituent of cigarette smoke, on cultured bovine retinal pigment epithelial (RPE) cells. Evidence is presented for its metabolic conversion into benzo[ a ]pyrene diol epoxide (BPDE) and the consequent formation of potentially cytotoxic nucleobase adducts in DNA. Cultured RPE cells were treated with BaP at concentrations in the range of 0–100 µm. The presence of BaP was found to cause inhibition of cell growth and replication. BaP induced the expression of a phase I drug metabolizing enzyme which was identified as cytochrome P450 1A1 (CYP 1A1) by RT–PCR and by Western blotting. Coincident with the increased expression of CYP 1A1, covalent adducts between the mutagenic metabolite BPDE and DNA could be detected within RPE cells by immunocytochemical staining. Additional support for their formation was afforded by nuclease P1 enhanced 32P-postlabelling assays on cellular DNA. Single-cell gel electrophoresis (comet) assays showed that exposure of RPE cells to BaP rendered them markedly more susceptible to DNA damage induced by broad band UVB or blue light laser irradiation. In the case of UVB, this is consistent with the photosensitization of DNA cleavage by nucleobase adducts of BPDE. Collectively, these findings imply that BaP has a significant impact on RPE cell pathophysiology and suggest mechanisms whereby exposure to cigarette smoke might cause RPE dysfunction and cell death, thus possibly contributing to degenerative disorders of the retina.

Terminal nerve-derived neuropeptide Y modulates physiological responses in the olfactory epithelium of hungry axolotls (Ambystoma mexicanum).

The vertebrate brain actively regulates incoming sensory information, effectively filtering input and focusing attention toward environmental stimuli that are most relevant to the animal's behavioral context or physiological state. Such centrifugal modulation has been shown to play an important role in processing in the retina and cochlea, but has received relatively little attention in olfaction. The terminal nerve, a cranial nerve that extends underneath the lamina propria surrounding the olfactory epithelium, displays anatomical and neurochemical characteristics that suggest that it modulates activity in the olfactory epithelium. Using immunocytochemical techniques, we demonstrate that neuropeptide Y (NPY) is abundantly present in the terminal nerve in the axolotl (Ambystoma mexicanum), an aquatic salamander. Because NPY plays an important role in regulating appetite and hunger in many vertebrates, we investigated the possibility that NPY modulates activity in the olfactory epithelium in relation to the animal's hunger level. We therefore characterized the full-length NPY gene from axolotls to enable synthesis of authentic axolotl NPY for use in electrophysiological experiments. We find that axolotl NPY modulates olfactory epithelial responses evoked by L-glutamic acid, a food-related odorant, but only in hungry animals. Similarly, whole-cell patch-clamp recordings demonstrate that bath application of axolotl NPY enhances the magnitude of a tetrodotoxin-sensitive inward current, but only in hungry animals. These results suggest that expression or activity of NPY receptors in the olfactory epithelium may change with hunger level, and that terminal nerve-derived peptides modulate activity in the olfactory epithelium in response to an animal's changing behavioral and physiological circumstances.

Expression of the 67 kDa laminin receptor (67LR) during retinal development: correlations with angiogenesis

Interaction of vascular cells with the laminin component of basement membranes is important for normal cell function. Likewise, abnormal interactions may have a critical role in vascular pathology. It has been previously demonstrated that the 67 kDa laminin receptor (67LR) is expressed at high levels during proliferative retinopathy in a mouse model and in the current study we have examined 67LR in the neonatal mouse to determine if this receptor plays a role in aspects of developmental angiogenesis in the developing murine retina. Groups of C57/BL6 mice were killed at postnatal day P1, P3, P5, P7, P9 and P11 to assess the retinal vasculature. A number of mice were perfused with FITC-dextran and the eyes removed, fixed in 4% paraformaldehyde (PFA) and flat-mounted for confocal scanning laser microscopy. The eyes from the remaining mice were either placed in 4% PFA and embedded in paraffin-wax, or had the neural retina dissected off and total RNA or protein extracted. Immunofluorescence, in situ hybridization, quantitative reverse transcriptase polymerase chain reaction and Western blotting analysis were employed to locate and determine expression levels of 67LR. Both 67LR mRNA and protein expression showed a characteristic bi-phasic expression pattern which correlated with key stages of retinal vascular development in the murine retina. 67LR showed high expression levels at P1 (P < 0.05) (correlating with superficial vascular plexus formation) and at P7 (P < 0.05) (correlating with deep vascular plexus formation). Conversely, 67LR expression was decreased when active angiogenic activity was lowest. Significantly, optical sectioning of retinal flat-mounts revealed high levels of 67LR expression in developing segments of both superficial and deep capillary plexi, a pattern which co-localized strongly with laminin. 67LR is regulated during post-natal development of the retinal vasculature. High levels of 67LR during the two well-defined phases of retinal capillary plexus formation suggests that this receptor may play an important role in retinal angiogenesis.