| 1. |
- Ali, Zaheer, et al.
(författare)
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Photoreceptor Degeneration Accompanies Vascular Changes in a Zebrafish Model of Diabetic Retinopathy
- 2020
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Ingår i: Investigative Ophthalmology and Visual Science. - : ASSOC RESEARCH VISION OPHTHALMOLOGY INC. - 0146-0404 .- 1552-5783. ; 61:2
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE. Diabetic retinopathy (DR) is a leading cause of vision impairment and blindness worldwide in the working-age population, and the incidence is rising. Until now it has been difficult to define initiating events and disease progression at the molecular level, as available diabetic rodent models do not present the full spectrum of neural and vascular pathologies. Zebrafish harboring a homozygous mutation in the pancreatic transcription factor pdx1 were previously shown to display a diabetic phenotype from larval stages through adulthood. In this study, pdx1 mutants were examined for retinal vascular and neuronal pathology to demonstrate suitability of these fish for modeling DR. METHODS. Vessel morphology was examined in pdx1 mutant and control fish expressing the fli1a:EGFP transgene. We further characterized vascular and retinal phenotypes in mutants and controls using immunohistochemistry, histology, and electron microscopy. Retinal function was assessed using electroretinography. RESULTS. Pdx1 mutants exhibit clear vascular phenotypes at 2 months of age, and disease progression, including arterial vasculopenia, capillary tortuosity, and hypersprouting, could be detected at stages extending over more than 1 year. Neural-retinal pathologies are consistent with photoreceptor dysfunction and loss, but do not progress to blindness. CONCLUSIONS. This study highlights pdx1 mutant zebrafish as a valuable complement to rodent and other mammalian models of DR, in particular for research into the mechanistic interplay of diabetes with vascular and neuroretinal disease. They are furthermore suited for molecular studies to identify new targets for treatment of early as well as late DR.
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| 2. |
- Blais, David R., et al.
(författare)
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LBP and CD14 secreted in tears by the lacrimal glands modulate the LPS response of corneal epithelial cells
- 2005
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Ingår i: Investigative Ophthalmology and Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 0146-0404 .- 1552-5783. ; 46:11, s. 4235-4244
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE. Lipopolysaccharide (LPS) is one of the most powerful bacterial virulence factors in terms of proinflammatory properties and is likely to contribute to corneal bacterial keratitis. Better understanding of the spatial expression of the LPS receptor components at the tear - corneal interface might facilitate enhanced functions of the LPS receptor complex in ocular defense against Gram-negative infections. METHODS. The expression of LPS-binding protein (LBP), CD14, toll-like receptor (TLR)-4, and MD-2 in human lacrimal glands, reflex tears, and corneal epithelia was examined by ELISA, RT-PCR, Western blot analysis, and immunofluorescence. The release of proinflammatory cytokines after the activation of primary and immortalized corneal epithelial cells with LPS and human tears was measured by ELISA. RESULTS. LBP and CD14 proteins were detected in reflex human tears. Human lacrimal glands and corneal epithelia expressed LBP, CD14, TLR4, and MD-2 mRNAs and proteins. In the corneal epithelium, LBP was mainly expressed by superficial and basal epithelial cells, whereas CD14, TLR4, and MD-2 expression were limited to the wing and basal epithelial cells. In a dose-dependant manner, tear CD14 and LBP mediated the secretion of interleukin (IL)-6 and IL-8 by corneal epithelia cells when challenged with LPS. CONCLUSIONS. Tear CD14 and LBP complemented the LPS receptor complex expressed by the corneal epithelia to trigger an immune response in the presence of LPS. The complementation of these tear and corneal immune proteins could play an important role in LPS recognition and signaling and, therefore, could modulate ocular innate immunity.
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| 3. |
- Bourghardt Peebo, Beatrice, et al.
(författare)
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Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy
- 2010
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Ingår i: Investigative Ophthalmology and Visual Science. - Rockville, MD, United States : Association for Research in Vision and Ophthalmology (ARVO). - 0146-0404 .- 1552-5783. ; 51:2, s. 830-835
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE. To determine whether in vivo confocal microscopy (IVCM) of the cornea can be used for the label-free detection and monitoring of lymph vessels in live corneas.METHODS. Parallel corneal hemangiogenesis and lymphangiogenesis was induced by the placement of a single suture in one cornea of male Wistar rats. Fourteen days after suture placement and under general anesthesia, laser-scanning IVCM was performed in the vascularized region. Corneas were subsequently excised for flat-mount double immunofluorescence with a pan-endothelial marker (PECAM-1/CD31) and a lymphatic endothelial specific marker (LYVE-1). Using the suture area and prominent blood vessels as points of reference, the identical microscopic region was located in both fluorescent and archived in vivo images. Additionally, vessel diameter, lumen contrast, and cell diameter and velocity within vessels were quantified from in vivo images.RESULTS. Comparison of identical corneal regions in fluorescence and in vivo revealed prominent CD31(+)/LYVE-1(3+) lymph vessels that were visible in vivo. In vivo, corneal lymph vessels were located in the vascularized area in the same focal plane as blood vessels but had a darker lumen (P andlt; 0.001) sparsely populated by highly reflective cells with diameters similar to those of leukocytes in blood vessels (P = 0.61). Cell velocity in lymph vessels was significantly reduced compared with blood particle velocity (P andlt; 0.001). Morphologic characteristics enabled subsequent identification of corneal lymphatics in live, vascularized rat corneas before immunofluorescence labeling.CONCLUSIONS. IVCM enabled the nondestructive, label-free, in vivo detection of corneal lymphatics. IVCM provides the possibility of observing lymphatic activity in the same live corneas longitudinally and, as a clinical instrument, of monitoring corneal lymphatics in live human subjects.
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| 4. |
- Bourghardt Peebo, Beatrice, et al.
(författare)
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Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting
- 2011
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Ingår i: Investigative Ophthalmology and Visual Science. - : Research in Vision and Opthalmology. - 0146-0404 .- 1552-5783. ; 52:6, s. 3060-3068
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE. To elucidate the temporal sequence of events leading to new capillary sprouting in inflammatory corneal angiogenesis. METHODS. Angiogenesis was induced by corneal suture placement in Wistar rats. The inflamed region was examined by time-lapse in vivo confocal microscopy for up to 7 days. At 6 and 12 hours and 1, 2, 4, and 7 days, corneas were excised for flat mount immunofluorescence with primary antibodies for CD31, CD34, CD45, CD11b, CD11c, Ki-M2R, NG2, and alpha-SMA. From days 0 to 4, the in vivo extravasation and expansion characteristics of single limbal vessels were quantified. RESULTS. Starting hours after induction and peaking at day 1, CD45(+)CD11b(+) myeloid cells extravasated from limbal vessels and formed endothelium-free tunnels within the stroma en route to the inflammatory stimulus. Limbal vessel diameter tripled on days 2 to 3 as vascular buds emerged and transformed into perfused capillary sprouts less than 1 day later. A subset of spindle-shaped CD11b(+) myeloid-lineage cells, but not dendritic cells or mature macrophages, appeared to directly facilitate further capillary sprout growth. These cells incorporated into vascular endothelium near the sprout tip, co-expressing endothelial marker CD31. Sprouts had perfusion characteristics distinct from feeder vessels and many sprout tips were open-ended. CONCLUSIONS. Time-lapse in vivo corneal confocal microscopy can be used to track a temporal sequence of events in corneal angiogenesis. The technique has revealed potential roles for myeloid cells in promoting vessel sprouting in an inflammatory corneal setting.
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