Mapping PERIOD-immunoreactive cells with neurons relevant to photoperiodic response in the bean bug Riptortus pedestris.

Circadian clock genes are involved in photoperiodic responses in many insects; however, there is a lack of understanding in the neural pathways that process photoperiodic information involving circadian clock cells. PERIOD-immunohistochemistry was conducted in the bean bug Riptortus pedestris to localise clock cells and their anatomical relationship with other brain neurons necessary for the photoperiodic response. PERIOD-immunoreactive cells were found in the six brain regions. In the optic lobe, two cell groups called lateral neuron lateral (LNl) and lateral neuron medial (LNm), were labelled anterior medial to the medulla and lobula, respectively. In the protocerebrum of the central brain, dorsal neuron (Prd), posterior neuron (Prp), and antennal lobe posterior neuron (pAL) were found. In the deutocerebrum, antennal lobe local neurons (ALln) were detected. Double immunohistochemistry revealed that PERIOD and serotonin were not co-localised. Furthermore, pigment-dispersing factor-immunoreactive neurons and anterior lobula neurons essential for R. pedestris photoperiodic response were not PERIOD immunopositive. LNl cells were located in the vicinity of the pigment-dispersing factor immunoreactive cells at the anterior base of the medulla. LNm cells were located close to the somata of the anterior lobula neurons. Fibres from the anterior lobula neurons and pigment-dispersing factor-immunoreactive neurons had contacts at the anterior base of the medulla. It is suggested that LNl cells work as clock cells involved in the photoperiodic response and the region at the medulla anterior base serves as a hub to receive photic and clock information relevant to the photoperiodic clock in R. pedestris.

Influence of the difference of breastfeeding volume on a rat model of oxygen-induced retinopathy.

This study aimed to investigate the effects of abundant breast milk intake on rats model of oxygen-induced retinopathy (OIR). Neonatal Sprague-Dawley rats were randomly assigned to expand litters of 7 pups/litter (7-rats group) and 14 pups/litter (14-rats group). They were exposed to 80% oxygen from postnatal day (P) 0 to P12. Body weights were measured daily. At P13 and 18, rats were sacrificed, and the blood and eyes were collected. Retinal neovascularization (NV) score, total retinal area (TRA), avascular area (AVA), and vascularized area (VA) were measured in ADPase stained retinas. Retinal vascular endothelial growth factor (VEGF) and serum insulin-like growth factor (IGF-1) were measured using ELISA. Body weight gain was significantly greater in 7-rats group from P2. Serum IGF-1 levels at P13 and 18 were significantly higher in 7-rats group. Retinal VEGF and TRA at P18 were significantly larger in 7-rats group. NV score at P18 tended to be higher in 7-rats group. There was no significant difference in VA between the 2 groups at P13 and 18. Excess breast milk intake in OIR rat pups caused body weight gain and retinal development, whereas there was less effect on retinal vascularization in our study.

ATYPICAL ENDOGENOUS FUNGAL ENDOPHTHALMITIS CAUSED BY CANDIDA RUGOSA.

PURPOSE: The purpose of this study was to report a case of atypical endogenous fungal endophthalmitis caused by Candida rugosa , a rare species of nonalbicans Candida . METHODS: This report describes a case of a 45-year-old woman who presented with a reduced visual acuity in the right eye in addition to vitreous opacity during breast cancer treatment, which was suspected as fungal endophthalmitis from medical examination and history. Various tests were performed for diagnosis. RESULTS: Blood test results were normal, including the blood beta-D-glucan level, and blood cultures were negative. Diagnosis could not be made using systemic computed tomography and magnetic resonance imaging results. Therefore, a lesion sample was collected by using vitrectomy. C. rugosa was identified through DNA (extracted from the lesion sample) analysis using Basic Local Alignment Search Tool. The visual acuity of the right eye improved after vitrectomy. CONCLUSION: We encountered a rare case of atypical endogenous fungal endophthalmitis caused by C. rugosa . Clinicians sometimes encounter invasive candidiasis caused by rare nonalbicans Candida species. DNA analysis using Basic Local Alignment Search Tool is effective for diagnosing such cases.

[Long term clinical outcomes of Baerveldt glaucoma implantation via the pars plana].

PURPOSE: To present the long-term follow-up results of Baerveldt glaucoma implant (BGI) with a drainage tube from the pars plana in secondary glaucoma patients. METHODS: The subjects were patients with light perception and secondary glaucoma (> 21 mmHg) who had received more than 2 glaucoma operations: 11 were neovascular glaucoma patients and 5 angle-closure glaucoma patients with peripheral anterior synechia. After vitreous surgery, a BGI plate was fixed at the sclera in the superior temporal equator, and a drainage tube inserted from the pars plana into the vitreous cavity. The successful criteria needed to satisfy the 3 following conditions: 1) vision of more than light perception, 2) IOP > or = 5 mmHg and not higher than 22 mmHg, 3) no need for further glaucoma operations. RESULTS: The follow-up period was 82.5 months (from 5 to 172 months). Eight patients were judged a success, 4 patients a failure and 4 patients quit for personal reasons. The 10-year success rate was estimated at 72.8%. CONCLUSIONS: BGI via the pars plana is a useful method for long term IOP lowering effect in secondary glaucoma.

Periostin, discovered by nano-flow liquid chromatography and mass spectrometry, is a novel marker of diabetic retinopathy.

Diabetes can lead to serious microvascular complications including proliferative diabetic retinopathy (PDR), the leading cause of blindness in adults. Recent studies using gene array technology have attempted to apply a hypothesis-generating approach to elucidate the pathogenesis of PDR, but these studies rely on mRNA differences, which may or may not be related to significant biological processes. To better understand the basic mechanisms of PDR and to identify potential new biomarkers, we performed shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS) analysis on pooled protein extracts from neovascular membranes obtained from PDR specimens and compared the results with those from non-vascular epiretinal membrane (ERM) specimens. We detected 226 distinct proteins in neovascular membranes and 154 in ERM. Among these proteins, 102 were specific to neovascular membranes and 30 were specific to ERM. We identified a candidate marker, periostin, as well as several known PDR markers such as pigment epithelium-derived factor (PEDF). We then performed RT-PCR using these markers. The expression of periostin was significantly up-regulated in proliferative membrane specimens. Periostin induces cell attachment and spreading and plays a role in cell adhesion. Proteomic analysis by LC/MS/MS, which permits accurate quantitative comparison, was useful in identifying new candidates such as periostin potentially involved in the pathogenesis of PDR.

Preserved and unpreserved 12 anti-allergic ophthalmic solutions and ocular surface toxicity: in vitro assessment in four cultured corneal and conjunctival epithelial cell lines.

In the present study, we evaluated the cytotoxicity of anti-allergic ophthalmic solutions in cultured corneal and conjunctival cells, namely SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). The viability of cell cultures was determined following the exposure of cells to 12 commercially available anti-allergic ophthalmic solutions for varying exposure times and at various dilutions using the MTT and neutral red assays. The cell viability score (CVS) was used to compare the toxicity of different drugs. Based on CVS data, the order of cell viability after exposure to the drugs was Zepelin ≥ Tramelas PF ≥ Cumorol PF ≥ Ketotifen PF ≥ Eyevinal = Fumarton ≥ Cumorol > Intal ≥ Rizaben ≥ Tramelas ≥ Patanol Livostin. In conclusion, cell viability was mostly affected by the concentration of benzalkonium chloride rather than the active component and/or the anti-allergic action of the drug. The CVS was useful in comparing the toxicity of different drugs.

Cytotoxicity of topical medications used for infection and inflammation control after cataract surgery in cultured corneal endothelial cells.

Postoperative vision-threatening corneal edema sometimes occurs after eye surgery, and corneal endothelial damage may be caused or exacerbated by drug toxicity. A range of commercially available antibiotic and anti-inflammatory ophthalmic solutions used postoperatively, namely levofloxacin, moxifloxacin, gatifloxacin, cefmenoxime, diclofenac, bromfenac, pranoprofen, betamethasone, and fluoromethorone, were assessed by using human corneal endothelial cells (HCECs). Propylparaoxybenzoate and methylparaoxybenzoate were also examined. Cell survival after 48 h exposure to the drugs was evaluated using the WST assay. Cefmenoxime and betamethasone were the least toxic antibiotic and anti-inflammatory drug, respectively. Cell survival was concentration dependent and increased markedly to ≥ 80% with dilutions of 100-fold or more. Two preservatives seemed to cause minimal cytotoxicity among those tested. Antibiotic cytotoxicity to HCEC was ranked as cefmenoxime < levofloxacin = gatifloxacin < moxifloxacin, while the toxicity of anti-inflammatory drugs was dependent on benzalkonium chloride and polysorbate. These drugs are unlikely to cause HCEC damage at the concentrations used under the usual conditions. Preservatives are essential ingredients in ophthalmic solutions to control postoperative infection and inflammation and we should be aware of their toxicity as well as efficacy.

Eye damage control by reduced blue illumination.

The aim of this study was to demonstrate that a blue light and ultraviolet cut-off filter (blue filter) could reduce short-wavelength retina/RPE damage threshold by a continuous spectrum source. Sixteen normal eyes of two rhesus monkeys and six cynomolgus monkeys were subjected to macular irradiation of 20, 24, 27.4, 30, 35, 45, 50 and 60 J/cm(2) energy densities. The values of energy density were measured before the blue filter. Lesions were measured before and at 2 and 30 days after irradiation of a 2.8 mm diameter region within the macular arcade. Measures were fundoscopy, fluorescein angiography and long wavelength scanning by the Heidelberg Retinal Tomograph (HRT) unit. The lesions, which were produced, were scored and compared to irradiant energy density of the blue LED (NSPB500S, Nichia, Tokushima, Japan). The exposure at the 20 J/cm(2) produced no detectable result at 2 or 30 days. Exposure at 35 J/cm(2) showed definite lesion production without blue filter. With the filter added there was one indication of minor change. At 60 J/cm(2) there was extensive heavy, enduring damage without the filter and with the filter damage was present but was significantly attenuated. These results strongly support the conclusion that the blue filter attenuation reduces the frequency of damage by exposure. This experimental system is a useful model for normal human eye aging and continuous spectrum environment irradiance.

[Cytotoxicity of antiglaucoma ophthalmic solutions for human corneal endothelial cells].

PURPOSE: The cytotoxicity of a range of commercial antiglaucoma ophthalmic solutions was assessed in human corneal endothelial cells using in vitro techniques. METHODS: Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, latanoplast, unoprostone, levobunolol, bunazosine, betaxolol, nipradiol, dorzolamide, brinzolamide, and pilocarpine were assessed. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 hours exposure. RESULTS: Survival was lower in prostagrandines and in medications containing benzalkonium. It increased to more than 85% after dilution of 1000-fold or more dilution. CONCLUSIONS: Antiglaucoma ophthalmic solutions have corneal endothelial toxicity. The toxicity significantly decreases after dilution of 1000-fold or more dilution and toxicity seems to be due mostly to benzalkonium chloride.

Cytotoxicity of ophthalmic solutions with and without preservatives to human corneal endothelial cells, epithelial cells and conjunctival epithelial cells.

PURPOSE: The cytotoxicity of a range of commercial ophthalmic solutions in the presence and absence of preservatives was assessed in human corneal endothelial cells (HCECs), corneal epithelia and conjunctival epithelia using in vitro techniques. METHODS: Cell survival was measured using the WST-1 assay for endothelial cells and the MTT assay for epithelial cells. Commercially available timolol, carteolol, cromoglicate, diclofenac, bromfenac and hyaluronic acid ophthalmic solutions were assessed for cytotoxicity in the presence and absence of preservatives. The preservatives benzalkonium, chlorobutanol and polysorbate were also tested. The survival of cells exposed to test ophthalmic solutions was expressed as a percentage of cell survival in the control solution (distilled water added to media) after 48 h exposure. RESULTS: HCEC survival was 20-30% in ophthalmic solutions diluted 10-fold. The survival of HCEC was significantly greater in all solutions in the absence of preservative than in the presence of preservative. The survival of corneal and conjunctival epithelia was consistent with that of HCECs for all test ophthalmic solutions. The preservatives polysorbate and benzalkonium were highly cytotoxic with cell survival decreasing to 20% at the concentration estimated in commercial ophthalmic solutions. By comparison, the survival of cells exposed to chlorobutanol was 80% or greater. CONCLUSIONS: The cytotoxicity of ophthalmic solutions to HCEC, corneal epithelia and conjunctival epithelia decreased in the absence of preservative.

[The effect of theanine on oxygen-induced retinal neovascularization in the neonatal rat].

PURPOSE: This study was aimed to investigate the preventive effects of theanine treatment on a rat model of oxygen-induced ischemic retinopathy (OIR). METHODS: OIR was induced by maintaining the Sprague-Dawley neonatal rats in 80% oxygen. The rats were treated once daily with gastric gavage of theanine (5 or 50 mg/kg) or distilled water (DW) from postnatal days 6 to 17. The retinal neovascularization (NV) was scored and avascular areas(AVAs) were measured as a % of total retinal area (% AVAs) at day 18. RESULTS: The % AVAs in 5 mg/kg theanine (13.2 +/- 2.8%) and 50 mg/kg theanine (9.4 +/- 2.2%, p < 0.05) treatment were lower than those in DW (18.9 +/- 2.9 %). The NV scores with 5 mg/kg theanine(4.2 +/- 0.5) or 50 mg/kg theanine (3.4 +/- 0.6) treatment were lower than those with DW (4.7 +/- 0.6). CONCLUSION: Theanine treatment suppresses the neovascularization in a rat model of OIR. These results suggest that theanine may prevent retinopathy of prematurity.

Negative Regulation of the Keap1-Nrf2 Pathway by a p62/Sqstm1 Splicing Variant.

A key antioxidant pathway, the Keap1-Nrf2 system, is regulated by p62/Sqstm1 via multiple mechanisms, including gene expression, posttranslational modifications (such as ubiquitination and phosphorylation), and autophagic degradation of p62/Sqstm1 and Keap1. Here we demonstrate a novel mode of regulation of the Keap1-Nrf2 system, mediated by a splicing variant of p62/Sqstm1 pre-mRNA. Ensembl database searches and subsequent biochemical analyses of mice revealed the presence of an mRNA that encodes a p62/Sqstm1 protein lacking the Keap1-interacting region (KIR), which is essential for the interaction with Keap1. Like full-length p62, the variant was induced under conditions in which Nrf2 was activated (e.g., impairment of autophagy), formed oligomers with itself and/or the full-length protein, and was degraded by autophagy. However, the variant failed to interact with Keap1 and sequester it in variant-positive aggregates. Remarkably, while full-length p62 stabilized Nrf2 and induced the gene expression of Nrf2 targets, the variant increased the amount of Keap1 and enhanced ubiquitination of Nrf2, thereby suppressing the induction of Nrf2 targets. Hepatocytes isolated from genetically modified mice that express full-length p62, but not the variant, were susceptible to activation of Nrf2 in response to stress. Collectively, our results suggest that splicing of p62/Sqstm1 pre-mRNA negatively regulates the Keap1-Nrf2 pathway.

Effect of ophthalmic solution components on acrylic intraocular lenses.

PURPOSE: To investigate the effect of ophthalmic solution components on the surface of acrylic intraocular lenses (IOLs). SETTING: Department of Opthalmology, Showa University School of Medicine. METHODS: Measurement of the contact angles of ophthalmic solutions on 3 acrylic IOLs was performed. The solutions were diclofenac sodium (Diclod), bromfenac sodium (Bronuck), betamethasone phosphate (Rinderon), dibekacin sulfate (Panimycin), polysorbate 80 (Tween 20), benzalkonium chloride, chlorobutanol, methylparahydroxybenzoate, and propylparahydroxybenzoate. The IOLs were incubated at 35 degrees C for 2 weeks in undiluted ophthalmic solutions and in 1:10 dilutions of ophthalmic solution components. The IOLs were sectioned and observed by scanning electron microscopy. RESULTS: The contact angle of Diclod and Bronuck solutions was the smallest. The contact angle of Rinderon and Panimycin was similar to that of distilled water. Scanning electron microscopy examination of IOLs incubated in ophthalmic solution components showed intralenticular changes. The IOLs immersed in ophthalmic solutions did not show any change, even after extended incubation. CONCLUSION: The chemical components of ophthalmic solutions, such as surfactants and solvents, permeate acrylic IOLs, suggesting the potential for long-term adverse effects of eyedrops in pseudophakic eyes.

Effects of green tea fractions on oxygen-induced retinal neovascularization in the neonatal rat.

This study aimed to investigate the preventive effects of green tea fractions (GTFs) on rat model of oxygen-induced retinopathy (OIR). Neonatal Sprague-Dawley rats were exposed to daily cycles of 80% oxygen (20.5 h), ambient air (0.5 h), and progressive return to 80% oxygen (3 h) until postnatal day 12 (P12), then the rats were placed in ambient air until P18. The green tea was fractionated by DM-A50, DM-W, M-B, and M-W. The rats were treated once daily from P6 to P17 by gastric gavage of GTFs (0.05 or 0.01 g/ml) or distilled water (DW) at 50 microl/10 g body weight. On P18, the rats were sacrificed and the retinal samples were collected. The retinal neovascularization (NV) was scored and avascular areas (AVAs) were measured as a % of total retinal area (%AVAs) in ADPase stained retinas. The NV scores in 0.01 g/ml M-W were significantly lower than those in DW. The %AVAs in 0.05 g/ml DM-A50 and in 0.05 g/ml and 0.01 g/ml M-W were significantly lower than those in DW. There were less catechins, and less caffeine in M-W fraction compared with other GTFs, suggesting components of green tea except for catechins and caffeine might suppress the neovascularization in rat model of OIR.

Activation of AP-1 and increased synthesis of MMP-9 in the rabbit retina induced by lipid hydroperoxide.

PURPOSE: We identified the temporal expression of activator protein-1 (AP-1) and matrix metalloproteinases (MMPs) after linoleic acid hydroperoxide (LHP) induction of retinal neovascularization. METHODS: After injection of LHP into the vitreous of rabbits, samples were collected for AP-1 binding activity and mRNA for MMP-9 and MMPs activity. AP-1 binding activity was measured by electrophoretic mobility shift assay. MMP-9 activity was measured by zymography and mRNA by quantitative RT-PCR. RESULTS: AP-1 binding activity was increased at 1-3 hr. MMP-9 mRNA levels were increased at 3 hr in the neural retina and by 12 hr in the retinal pigment epithelium (RPE) layer. MMP-9 proteolytic activity was elevated within the neural retina and within the vitreous and in the RPE-interphotoreceptor matrix (IPM) at 12 hr and peaked at 24 hr or 4 days. CONCLUSIONS: LHP increases the transcription factor AP-1 which in turn may regulate retinal MMP-9 synthesis during neovascularization.

Surfactant induced glistenings: surface active ingredients in ophthalmic solutions may enhance water entry into the voids of implanted acrylic intraocular lenses.

PURPOSE: To investigate the light scattering properties of acrylic intraocular lenses (IOLs) placed in eyes treated with diclofenac sodium (Diclod, Wakamoto, Japan) and betamethasone phosphate (Rinderon, Shionogi, Japan) ophthalmic solutions. METHODS: The light scattering properties of IOLs (MA30BA, Alcon laboratories) placed in 32 eyes treated with Diclod and 31 treated with Rinderon were evaluated 3 years after implantation using Scheimpflug photography. RESULTS: The averaged intensity of glistenings was 9.0 CCT (computer compatible tapes) in the Diclod test group and 4.2 CCT in the Rinderon test group (p = 0.002). The intensity of surface light scattering for the Diclod and Rinderon test groups was 24.0 CCT and 26.9 CCT, respectively (p = 0.081). CONCLUSION: The Diclod test group had a greater number of glistenings than the Rinderon test group. Both drugs have similar anti-inflammatory properties and Diclod, unlike Rinderon, also contains the surfactant polysorbate. We proposed that the presence of surfactant, or other ingredient, in commercially available eyedrops may enhance the development of glistenings.

[The effect of Sairei-to on oxygen-induced retinal neovascularization in the neonatal rat].

PURPOSE: To study the effect of Sairei-to (ST), a Japanese traditional medicine, on oxygen-induced retinopathy (OIR) in rats. METHODS: OIR was induced by maintaining Sprague-Dawley neonatal rats in 80% oxygen for 12 days. The rats were treated once daily with oral administration of 0.75 g/kg (n = 9), 1.5 g/kg (n = 13) of ST in water, or water alone (WA, n = 13) at 5 mL/ kg body weight from day 6 to day 17. On day 18, retinal samples were collected. Retinal neovascularization (NV) was assessed by the NV score, and by the percentage of avascular area (% AVA), using a method previously reported. The number of severe retinal NV cases (NV > or = 9) was compared. The retinal vascular endothelial growth factor (VEGF) concentrations were measured with an immunoassay kit, at 0, 12, 24, 72 and 144 hours after oxygenation. RESULTS: NV score and % AVA decreased in the ST treated group compared to the WA group. However, severe NV was seen in five cases of WA and in one case of the ST treated group. Thus severe NV was inhibited significantly by ST treatment (p = 0.0185). Retinal VEGF did not differ between groups at any time points. CONCLUSION: These data suggest that severe NV in OIR is inhibited by ST treatment.

Aryl Hydrocarbon Receptor Antagonist StemRegenin 1 Promotes the Expansion of Human Promyelocytic Leukemia Cell Line, NB4.

BACKGROUND/AIM: StemRegenin 1 (SR1), an antagonist of aryl hydrocarbon receptor (AHR), reportedly promotes expansion of hematopoietic stem cells but its effect on leukemia cells is unclear. This study focused on the role of SR1 in leukemia cell proliferation. MATERIALS AND METHODS: AHR expression was compared in the cell lines Jurkat, Kasumi-1, NB4 and K562, using real-time polymerase chain reaction. Highly AHR-expressing NB4 cells were cultured with SR1 for 2 and 4 days, and evaluated for viability and gene expression. DNA microarray was also performed. RESULTS: The viability of NB4 cells treated with 1.5 µM SR1 increased at day 4. Expression of B-cell CLL/lymphoma 2 (BCL2) was up-regulated, while that of BCL2 associated X protein (BAX) was down-regulated at day 2. Increased cyclin D1 (CCND1), CCND2 and v-myc avian myelocytomatosis viral oncogene homolog (MYC) expressions were observed at day 4. Global gene expression profiles showed up-regulation of splice variant-related genes and down-regulation of inflammation-related genes. CONCLUSION: SR1 promotes the expansion of NB4 cells in vitro, implying the need for caution regarding in vivo use of R1.

Lipid hydroperoxide induced corneal neovascularization in hyperglycemic rabbits.

PURPOSE: To compare the effect of hyperglycemia on corneal neovascularization (NV) induced by linoleic acid hydroperoxide (LHP) in a rabbit model. METHODS: Male New Zealand rabbits received 80 mg/kg alloxan i.v. and subsequently developed hyperglycemia. Four weeks later, 10 microl of LHP (40 mM) was injected into corneal stroma 5 mm from the superior limbus with a 30 gauge needle. Vessel growth area from the limbal vasculature was measured over a period of 2 weeks and was correlated with plasma levels of insulin, HbA(1c), and corneal vascular endothelial growth factor (VEGF). RESULTS: Two days after alloxan, blood glucose was increased from 97 +/- 4 mg/dl in the untreated control group to 413 +/- 3 mg/dl. At 24 and 72 hours after LHP injection, VEGF in cornea of hyperglycemic rabbits was elevated 2 to 4 times above that of normoglycemic rabbits. At 14 days after LHP injection, the normoglycemic rabbits vessel growth area measured 2.42 +/- 0.31 mm(2), but in the hyperglycemic group, vessel growth area was significantly increased to 7.96 +/- 2.26 mm(2) (p < 0.05). At the end of the experimental period, HbA(1c) was elevated from 3.9 +/- 0.8 % to 8.4 +/- 0.6 % and insulin was decreased from 440 +/- 123.9 pg/ml to 24 +/- 11.0 pg/ml. CONCLUSIONS: These data suggest that hyperglycemia may sensitize corneal and vascular endothelial cells, perhaps by glucose derived radicals, which enhance production of additional LHP through endogenous propagation reactions, and raise in turn the concentration of VEGF levels to induce an enhanced, sustained NV response.