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- 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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| 2. |
- 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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| 5. |
- Germundsson, Johan, et al.
(författare)
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Age-Related Thinning of Bowman's Layer in the Human Cornea In Vivo
- 2013
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Ingår i: Investigative Ophthalmology and Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 0146-0404 .- 1552-5783. ; 54:9, s. 6143-6149
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Tidskriftsartikel (refereegranskat)abstract
- Purpose. To determine the thickness of Bowman's layer (BL) in vivo in a healthy population and to determine its variation with age.Methods. Eighty-two subjects aged 15 to 88 years with clear, healthy corneas were examined bilaterally with laser scanning in vivo confocal microscopy (IVCM). Bowman's layer thickness was determined from IVCM images of anterior and posterior BL boundaries. For a given eye, BL thickness was averaged across four central locations by two independent observers. In addition, central corneal thickness was measured by time-domain optical coherence tomography.Results. A significant negative correlation of BL thickness with age was found in right eyes (Pearson r = −0.579, P < 0.0001) and in left eyes (r = −0.558, P < 0.0001). Linear regression analysis yielded a decline in BL thickness of 0.06 μm per year. In 41 older subjects (mean age, 64.4 years), BL thickness was significantly thinner (mean ± SD, 8.6 ± 1.7 μm in right eyes) than that in 41 younger subjects (mean age, 31.6 years) (mean ± SD, 10.7 ± 1.6 μm in right eyes) (P < 0.001). No correlation of corneal thickness with age or of BL thickness with corneal thickness was observed. Strong intereye correlations in BL thickness (r = 0.771, P < 0.0001) and corneal thickness (r = 0.969, P < 0.001) were found.Conclusions. Bowman's layer thins with age in the normal cornea, losing one-third of its thickness between the ages of 20 and 80 years. In vivo measurement of BL thickness by IVCM could aid in clinical assessment and planned treatments of the anterior cornea.
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| 6. |
- Germundsson, Johan, et al.
(författare)
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An Accurate Method to Determine Bowmans Layer Thickness In Vivo in the Human Cornea
- 2012
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Ingår i: Investigative Ophthalmology and Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 0146-0404 .- 1552-5783. ; 53:4, s. 2354-2359
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE. To determine an accurate value for Bowmans layer (BL) thickness in vivo in humans. less thanbrgreater than less thanbrgreater thanMETHODS. Seventeen corneal transplant patients were examined preoperatively by laser-scanning in vivo confocal microscopy (IVCM), and corneal buttons were removed post-operatively and sectioned for light microscopy (LM). Nine corneas with uniformly thick BL by LM were used for thickness measurement. In the uniformly thick samples, probable overestimation of BL thickness in vivo by a first in vivo method (Method 1) led to the development of a revised in vivo method (Method 2). Method 2 was used to measure BL thickness in 20 healthy volunteers. less thanbrgreater than less thanbrgreater thanRESULTS. In nine patients, mean BL thickness prior to transplantation was 13.7 +/- 1.6 mu m by IVCM (Method 1) while BL thickness of the removed corneal button was 9.7 +/- 1.7 mu m by LM (P andlt; 0.001). The correlation of BL thickness between IVCM (Method 1) and LM was poor (P = 0.226). In 20 right eyes of 20 normal corneas, both in vivo methods were used to determine BL thickness. Mean BL thickness by Method 1 was 13.2 +/- 1.6 mu m and by Method 2 was 9.1 +/- 1.4 mu m (P andlt; 0.001). BL thickness measurements by both in vivo methods were highly correlated (P andlt; 0.001). less thanbrgreater than less thanbrgreater thanCONCLUSION. BL thickness by a revised in vivo method was close to LM values in this study and to values reported in fixed tissue in other studies. The authors believe this revised method provides the most accurate estimates of BL thickness in vivo to date.
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| 7. |
- Hackett, Joanne M., et al.
(författare)
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Biosynthetic corneal implants for replacement of pathologic corneal tissue : performance in a controlled rabbit alkali burn model
- 2011
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Ingår i: Investigative Ophthalmology and Visual Science. - : Research in Vision and Opthalmology. - 0146-0404 .- 1552-5783. ; 52:2, s. 651-657
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: To evaluate the performance of structurally reinforced, stabilized recombinant human collagen-phosphorylcholine (RHCIII-MPC) hydrogels as corneal substitutes in a rabbit model of severe corneal damage. Methods: One eye each of 12 rabbits received a deep corneal alkali wound. Four corneas were implanted with RHCIII-MPC hydrogels. The other eight control corneas were implanted with either allografts or a simple crosslinked RHCIII hydrogel. In all cases, 6.25 mm diameter, 350 µm thick buttons were implanted by anterior lamellar keratoplasty to replace damaged corneal tissue. Implants were followed for nine months by clinical examination and in vivo confocal microscopy, after which implanted corneas were removed and processed for histopathological and ultrastructural examination. Results: Alkali exposure induced extensive central corneal scarring, ocular surface irregularity, and neovascularization in one case. All implants showed complete epithelial coverage by four weeks post-operative, but with accompanying suture-induced vascularization in 6/12 cases. A stable, stratified epithelium with hemidesmosomal adhesion complexes regenerated over all implants, and subbasal nerve regeneration was observed in allograft and RHCIII-MPC implants. Initially acellular biosynthetic implants were populated with host-derived keratocytes as stromal haze subsided and stromal collagen was remodeled. Notably, RHCIII-MPC implants exhibited resistance to vascular ingrowth while supporting endogenous cell and nerve repopulation. Conclusion: Biosynthetic implants based on RHC promoted cell and nerve repopulation in alkali burned rabbit eyes. In RHCIII-MPC implants, evidence of an enhanced resistance to neovascularization was additionally noted.
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| 9. |
- Lagali, Neil, et al.
(författare)
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Donor and recipient endothelial cell population of the transplanted human cornea: a two-dimensional imaging study.
- 2010
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Ingår i: Investigative ophthalmology & visual science. - : Association for Research in Vision and Ophthalmology (ARVO). - 1552-5783 .- 0146-0404. ; 51:4, s. 1898-904
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Tidskriftsartikel (refereegranskat)abstract
- Purpose. To elucidate the pattern of donor and recipient endothelial cell populations in transplanted human corneas and determine the degree to which donor endothelial cells survive in the graft. Methods. Thirty-six corneal grafts were collected from recipients of opposite sex to the donor, at the time of retransplantation for various indications. Cells from the endothelial side of the grafts were harvested, preserving their relative location on the endothelium. Fluorescence in situ hybridization of the sex chromosomes enabled each cell to be identified as donor- or recipient-derived. Images of the graft endothelium were assembled, to depict the pattern of cell population of the graft, and the proportion of donor cells present was estimated. Results. Endothelial cells of donor origin were found in 26 of 36 grafts (72.2%)-in one case, up to 26 years after transplantation. The proportion of donor endothelium ranged from 2% to 99%; however, there was no significant correlation of this proportion with postoperative time (P = 0.19). The mean annual rate of donor cell loss correlated negatively with the time to graft failure by endothelial decompensation (P = 0.002). Endothelial images indicated a highly variable pattern of recipient cell repopulation of the graft. A tendency toward donor cell retention in transparent, successful grafts was noted; however, this feature alone was not a reliable indicator of long-term graft transparency. Conclusions. Two-dimensional imaging of the corneal graft endothelium revealed a variable pattern and extent of donor and recipient cell population, indicating the highly dynamic nature of the corneal endothelium after transplantation.
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| 10. |
- Lagali, Neil, et al.
(författare)
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In Vivo Morphology of the Limbal Palisades of Vogt Correlates With Progressive Stem Cell Deficiency in Aniridia-Related Keratopathy
- 2013
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Ingår i: Investigative Ophthalmology and Visual Science. - : Association for Research in Vision and Ophthalmology (ARVO). - 0146-0404 .- 1552-5783. ; 54:8, s. 5333-5342
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Tidskriftsartikel (refereegranskat)abstract
- Purpose. To investigate morphologic alterations in the limbal palisades of Vogt in a progressive form of limbal stem cell deficiency.Methods. Twenty Norwegian subjects (40 eyes) with congenital aniridia and 9 healthy family members (18 eyes) without aniridia were examined. Clinical grade of aniridia-related keratopathy (ARK) was assessed by slit-lamp biomicroscopy, and tear production and quality, corneal thickness, and sensitivity were additionally measured. The superior and inferior limbal palisades of Vogt and central cornea were examined by laser scanning in vivo confocal microscopy (IVCM).Results. In an aniridia patient with grade 0 ARK, a transparent cornea and normal limbal palisade morphology were found. In grade 1 ARK, 5 of 12 eyes had degraded palisade structures. In the remaining grade 1 eyes and in all 20 eyes with stage 2, 3, and 4 ARK, palisade structures were absent by IVCM. Increasing ARK grade significantly correlated with reduced visual acuity and corneal sensitivity, increased corneal thickness, degree of degradation of superior and inferior palisade structures, reduced peripheral nerves, increased inflammatory cell invasion, and reduced density of basal epithelial cells and central subbasal nerves. Moreover, limbal basal epithelial cell density and central corneal subbasal nerve density were both significantly reduced in aniridia compared to healthy corneas (P = 0.002 and 0.003, respectively).Conclusions. Progression of limbal stem cell deficiency in aniridia correlates with degradation of palisade structures, gradual transformation of epithelial phenotype, onset of inflammation, and a corneal nerve deficit. IVCM can be useful in monitoring early- to late-stage degenerative changes in stem cell–deficient patients.
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