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  • 1.
    Bourghardt Peebo, Beatrice
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Angiogenesis from a new perspective2012Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Angiogenesis is the emergence of new blood and lymph vessels from existing ones. In the pathologic form it contributes to the onset and progression of numerous different human disorders such as cancer, inflammation, atherosclerosis and blinding eye diseases. There exist a number of models to study angiogenesis, both in vitro and in vivo, but there is no single perfect model so far. Consequently there is a need to develop new angiogenesis assays for evaluating blood and lymph vessel behaviour in different physiologic settings.

    The aim of this thesis was to gain insight into in vivo angiogenesis introducing a new technique in an inflammatory corneal model. The method involved in vivo examination of the cornea and subsequent comparison of in vivo findings with ex vivo immunohistochemical analysis of the same tissue samples. An existing suture model for inflammatory angiogenesis in the cornea was modified for in vivo observations with a clinically-approved corneal confocal microscope.

    In this thesis, corneal lymph vessels were characterized for the first time in vivo and findings from the experimental bench could be applied in a clinical setting, where presumed lymphatics were observed in a corneal transplant patient with rejection. Furthermore, the technique was extended to investigate time-lapse processes in sprouting and regressing capillaries, and led to a number of new observations. CD11b+ myeloid cells constitute the first bulk of infiltrating inflammatory cells and contribute to inflammatory sprouting and regression in numerous ways including pre-patterning of the corneal stroma and guiding of capillary sprouts. Newly formed hemangiogenic sprouts are perfused with a slow-moving fluid and have a lumen. In blood vessel regression, capillary remodeling occurred by abandonment of sprout tips in close association with macrophages and vascular loops formed by presumed intussusceptive angiogenesis. In addition, a network of pericyte- and endothelium-free basement membrane tubes was formed after desertion or degradation of vascular endothelium in former corneal capillaries.

    In conclusion, we introduce a new in vivo technique for investigating angiogenesis in a corneal model were in vivo findings can be interpreted with ex vivo definitions of specific cell types by immunohistochemistry. Findings from pre-clinical experiments have been possible to apply in a clinical setting when examining patients with corneal pathology.

    Delarbeid
    1. Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy
    Åpne denne publikasjonen i ny fane eller vindu >>Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy
    2010 (engelsk)Inngår i: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 51, nr 2, s. 830-835Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    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.

    sted, utgiver, år, opplag, sider
    Rockville, MD, United States: , 2010
    HSV kategori
    Identifikatorer
    urn:nbn:se:liu:diva-53820 (URN)10.1167/iovs.09-4407 (DOI)000273704700030 ()
    Tilgjengelig fra: 2010-02-05 Laget: 2010-02-05 Sist oppdatert: 2018-01-22bibliografisk kontrollert
    2. Letter: In vivo confocal microscopy visualization of presumed lymph vessels in a case of corneal transplant rejection
    Åpne denne publikasjonen i ny fane eller vindu >>Letter: In vivo confocal microscopy visualization of presumed lymph vessels in a case of corneal transplant rejection
    2011 (engelsk)Inngår i: Clinical and Experimental Ophthalmology, ISSN 1442-6404, E-ISSN 1442-9071, Vol. 39, nr 8, s. 832-834Artikkel i tidsskrift, Letter (Annet vitenskapelig) Published
    Abstract [en]

    n/a

    sted, utgiver, år, opplag, sider
    Wiley-Blackwell, 2011
    HSV kategori
    Identifikatorer
    urn:nbn:se:liu:diva-72259 (URN)10.1111/j.1442-9071.2011.02557.x (DOI)000296913900016 ()
    Tilgjengelig fra: 2011-11-24 Laget: 2011-11-24 Sist oppdatert: 2018-01-22bibliografisk kontrollert
    3. Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting
    Åpne denne publikasjonen i ny fane eller vindu >>Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting
    2011 (engelsk)Inngår i: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 52, nr 6, s. 3060-3068Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    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.

    sted, utgiver, år, opplag, sider
    Research in Vision and Opthalmology, 2011
    HSV kategori
    Identifikatorer
    urn:nbn:se:liu:diva-69178 (URN)10.1167/iovs.10-6101 (DOI)000291100800026 ()
    Merknad
    Original Publication: Beatrice Bourghardt Peebo, Per Fagerholm, Catharina Traneus-Rockert and Neil Lagali, Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting, 2011, INVESTIGATIVE OPHTHALMOLOGY and VISUAL SCIENCE, (52), 6, 3060-3068. http://dx.doi.org/10.1167/iovs.10-6101 Copyright: Research in Vision and Opthalmology http://www.arvo.org/Tilgjengelig fra: 2011-06-17 Laget: 2011-06-17 Sist oppdatert: 2018-01-22bibliografisk kontrollert
    4. Cellular level characterization of capillary regression in inflammatory angiogenesis using an in vivo corneal model
    Åpne denne publikasjonen i ny fane eller vindu >>Cellular level characterization of capillary regression in inflammatory angiogenesis using an in vivo corneal model
    2011 (engelsk)Inngår i: Angiogenesis, ISSN 0969-6970, E-ISSN 1573-7209, Vol. 14, nr 3, s. 393-405Artikkel i tidsskrift (Fagfellevurdert) Published
    Abstract [en]

    In this study, we introduce a technique for repeated, microscopic observation of single regressing capillaries in vivo in inflamed murine corneas. Natural capillary regression was initiated by removal of inflammatory stimulus during an active pro-angiogenic phase, while the additional impact of anti-angiogenic treatment with triamcinolone or bevazicumab was investigated. Capillaries regressed naturally within 1 week and treatments did not further enhance the natural regression. Morphologically, early-phase regression was characterized by significant lumen narrowing and a significant reduction in CD11b+ myeloid cell infiltration of the extracellular matrix. By 1 week, vascular remodeling occurred concomitant with CD11b+CD68+KiM2R+ mature macrophage localization on capillary walls. Empty conduits without blood flow, positive for collagen IV and devoid of vascular endothelium and pericytes, were apparent in vivo and by 3 weeks were more numerous than perfused capillaries. By 3 weeks, macrophages aggregated around remaining perfused capillaries and were observed in apposition with degrading capillary segments. Abrupt termination of capillary sprouting in our regression model further revealed vascular endothelial abandonment of sprout tips and perfused capillary loop formation within a single angiogenic sprout, possibly as an intussusceptive response to cessation of the stimulus. Finally, we observed lumen constriction and macrophage localization on capillary walls in vivo in a clinical case of corneal capillary regression that paralleled findings in our murine model.

    sted, utgiver, år, opplag, sider
    Springer Verlag (Germany), 2011
    Emneord
    Inflammation, Capillary regression, In vivo confocal microscopy, Cornea
    HSV kategori
    Identifikatorer
    urn:nbn:se:liu:diva-70325 (URN)10.1007/s10456-011-9223-3 (DOI)000293922300015 ()
    Merknad
    The original publication is available at www.springerlink.com: Beatrice Bourghardt Peebo, Per Fagerholm, Catharina Traneus-Rockert and Neil Lagali, Cellular level characterization of capillary regression in inflammatory angiogenesis using an in vivo corneal model, 2011, Angiogenesis, (14), 3, 393-405. http://dx.doi.org/10.1007/s10456-011-9223-3 Copyright: Springer Verlag (Germany) http://www.springerlink.com/Tilgjengelig fra: 2011-09-02 Laget: 2011-09-02 Sist oppdatert: 2018-01-22
  • 2.
    Bourghardt Peebo, Beatrice
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Fagerholm, Per
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neurovetenskap. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Lagali, Neil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neurovetenskap. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    An in Vivo Method for Visualizing Flow Dynamics of Cells within Corneal Lymphatics2013Inngår i: Lymphatic Research and Biology, ISSN 1539-6851, E-ISSN 1557-8585, Vol. 11, nr 2, s. 93-100Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Background: Monitoring the trafficking of specific cell populations within lymphatics could improve our understanding of processes such as transplant rejection and cancer metastasis. Current methods, however, lack appropriate image resolution for single-cell analysis or are incompatible with in vivo and longitudinal monitoring of lymphatics in their native state. We therefore sought to achieve high-resolution live imaging of the dynamic behavior of cells within lymph vessels in the rat cornea.

    Methods/Results: Inflammatory angiogenesis was induced by suture placement in corneas of Wistar rats. Pre- and up to 3 weeks post-induction, corneas were noninvasively examined by laser-scanning in vivo corneal confocal microscopy (IVCM) using only endogenous contrast. Lymph vessels and the cells harbored therein were documented by still images, real-time video, and 3D confocal stack reconstruction of live tissue. In vivo, conjunctival and corneal lymphatics were morphologically distinct, those with corneal location being one-quarter the diameter of those in the conjunctiva (p<0.001). Cells were recruited to initially empty pre-existing lymph vessels during the first day of inflammation and maintained a dense occupation of vessels for up to 7 days. A diverse population of cells (diameter range: 1.5–27.5 μm) with varying morphology was observed, and exhibited variable flow patterns and were transported singly and in clusters of at least 2–9 adherent cells.

    Conclusions: The in vivo microscopic technique presented enables lymph vessels and cell trafficking to be studied in high resolution in a minimally-perturbed physiologic milieu.

  • 3.
    Bourghardt Peebo, Beatrice
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Fagerholm, Per
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Lagali, Neil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Letter: In vivo confocal microscopy visualization of presumed lymph vessels in a case of corneal transplant rejection2011Inngår i: Clinical and Experimental Ophthalmology, ISSN 1442-6404, E-ISSN 1442-9071, Vol. 39, nr 8, s. 832-834Artikkel i tidsskrift (Annet vitenskapelig)
    Abstract [en]

    n/a

  • 4.
    Bourghardt Peebo, Beatrice
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US/LiM.
    Fagerholm, Per
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US/LiM.
    Lagali, Neil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US/LiM.
    Transient Anterior Corneal Deposits in a Human Immunodeficiency Virus-Positive Patient2010Inngår i: CORNEA, ISSN 0277-3740, Vol. 29, nr 11, s. 1323-1327Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Purpose: To report findings of pigmented anterior corneal deposits in a human immunodeficiency virus-positive patient. Methods: Case report. A 49-year-old human immunodeficiency virus-positive patient was examined after the appearance of pigmented corneal deposits. Slit-lamp biomicroscopy, fundus photography, and laser-scanning in vivo confocal microscopy were performed to visually document the ocular condition. Results: The patient had a history of Mycobacterium avium infection and was suspected to have recovery uveitis from a cytomegalovirus infection. Small, rounded, light brown-colored deposits were distributed across the anterior cornea from limbus to limbus, bilaterally. In vivo confocal microscopy revealed the deposits to be confined to the basal epithelium and Bowman layer, whereas the posterior stroma, Descemet membrane, and the endothelium appeared normal. Systemic steroid treatment was administered, and 2 weeks later, the deposits had vanished on slit-lamp examination, whereas remnants were observed at the microscopic level. Conclusions: The deposits were unusual for their anterior corneal location and pancorneal distribution. The response to systemic steroid treatment remains unexplained and illustrates the complexity of the underlying conditions, their treatment, and the associated pathways of ocular manifestation.

  • 5.
    Bourghardt Peebo, Beatrice
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Fagerholm, Per
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Traneus-Rockert, Catharina
    Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk patologi och klinisk genetik.
    Lagali, Neil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Cellular level characterization of capillary regression in inflammatory angiogenesis using an in vivo corneal model2011Inngår i: Angiogenesis, ISSN 0969-6970, E-ISSN 1573-7209, Vol. 14, nr 3, s. 393-405Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In this study, we introduce a technique for repeated, microscopic observation of single regressing capillaries in vivo in inflamed murine corneas. Natural capillary regression was initiated by removal of inflammatory stimulus during an active pro-angiogenic phase, while the additional impact of anti-angiogenic treatment with triamcinolone or bevazicumab was investigated. Capillaries regressed naturally within 1 week and treatments did not further enhance the natural regression. Morphologically, early-phase regression was characterized by significant lumen narrowing and a significant reduction in CD11b+ myeloid cell infiltration of the extracellular matrix. By 1 week, vascular remodeling occurred concomitant with CD11b+CD68+KiM2R+ mature macrophage localization on capillary walls. Empty conduits without blood flow, positive for collagen IV and devoid of vascular endothelium and pericytes, were apparent in vivo and by 3 weeks were more numerous than perfused capillaries. By 3 weeks, macrophages aggregated around remaining perfused capillaries and were observed in apposition with degrading capillary segments. Abrupt termination of capillary sprouting in our regression model further revealed vascular endothelial abandonment of sprout tips and perfused capillary loop formation within a single angiogenic sprout, possibly as an intussusceptive response to cessation of the stimulus. Finally, we observed lumen constriction and macrophage localization on capillary walls in vivo in a clinical case of corneal capillary regression that paralleled findings in our murine model.

  • 6.
    Bourghardt Peebo, Beatrice
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US/LiM.
    Fagerholm, Per
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US/LiM.
    Traneus-Rockert, Catharina
    Östergötlands Läns Landsting, Laboratoriemedicinskt centrum, Klinisk patologi och klinisk genetik.
    Lagali, Neil
    Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US/LiM. Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet.
    Cellular-Level Characterization of Lymph Vessels in Live, Unlabeled Corneas by In Vivo Confocal Microscopy2010Inngår i: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 51, nr 2, s. 830-835Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 7.
    Bourghardt Peebo, Beatrice
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Fagerholm, Per
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Traneus-Rockert, Catharina
    Östergötlands Läns Landsting, Diagnostikcentrum, Klinisk patologi och klinisk genetik.
    Lagali, Neil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmiatrik. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Time-Lapse In Vivo Imaging of Corneal Angiogenesis: The Role of Inflammatory Cells in Capillary Sprouting2011Inngår i: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 52, nr 6, s. 3060-3068Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    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.

  • 8.
    Bourghardt Peebo, Beatrice
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan, Oftalmologi. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US.
    Gan, Lisha
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan, Oftalmologi.
    Sun, Xiao-Feng
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Onkologi. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Onkologiska kliniken US.
    Knutsen Holmqvist, Annica
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för biomedicin och kirurgi, Onkologi. Östergötlands Läns Landsting, Kirurgi- och onkologicentrum, Onkologiska kliniken US.
    Rearden, Ann
    Fagerholm, Per
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan, Oftalmologi. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US.
    Expression of the focal adhesion protein PINCH in normal and alkali-injured corneas and the role of PMNs2007Inngår i: Acta Ophthalmologica Scandinavica, ISSN 1395-3907, E-ISSN 1600-0420, Vol. 85, nr 4, s. 395-400Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Purpose: To evaluate the role of particularly interesting new cysteine-histidine-rich protein (PINCH) in corneal wound healing and early neovascularization and to assess the influence of granulocytes. Methods: A standardized corneal alkali wound was inflicted under general anaesthesia to the right eye of 14 New Zealand White rabbits. Seven of the rabbits received i.v. 5 mg/kg fucoidin every 2 hours to prevent granulocytes from entering the wound area. After 36 hours, the rabbits were killed, the corneas excised, fixed in 4% formaldehyde and embedded in paraffin. The sections were double-stained with antibodies against PINCH and with haematoxylin. Results: In the normal cornea and limbus, PINCH was weakly expressed in the corneal epithelium and in a wedge of the conjunctival stroma. In the wounded corneas, PINCH expression was seen in the frontline of repopulating endothelial and epithelial cells, and in active keratocytes. The vascular endothelium and the granulocytes expressed PINCH, as did the conjunctival epithelium. In the fucoidin-treated rabbits, PINCH expression was markedly reduced. The vascular endothelial cells and the few granulocytes did not express PINCH in these rabbits. Conclusions: PINCH is only slightly expressed in the normal cornea. A corneal wound induces PINCH expression in the repopulating cells, in the vascular endothelial cells of the limbus, in the limbal epithelium and in the granulocytes. Exclusion of granulocytes reduces expression of PINCH and there is no expression at all in the vascular endothelium. © 2007 The Authors Journal compilation 2007 Acta Ophthalmol Scand.

  • 9.
    Bourghardt Peebo, Beatrice
    et al.
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan, Oftalmologi. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US.
    Koulikovska, Marina
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan, Oftalmologi. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US.
    Fagerholm, Per
    Linköpings universitet, Hälsouniversitetet. Linköpings universitet, Institutionen för nervsystem och rörelseorgan, Oftalmologi. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US.
    The suppression of early angiogenic markers by the antiangiogenic aptamer Macugen R is dose dependent2007Inngår i: European Association for Vision and Eye Research,2007, 2007Konferansepaper (Annet vitenskapelig)
  • 10.
    Frennesson, Christina
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US/LiM.
    Nilsson, Ulla
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmologi. Linköpings universitet, Hälsouniversitetet.
    Bourghardt Peebo, Beatrice
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmologi. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US/LiM.
    Nilsson, Sven Erik
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Oftalmologi. Linköpings universitet, Hälsouniversitetet.
    Significant improvements in near vision, reading speed, central visual field and related quality of life after ranibizumab treatment of wet age-related macular degeneration2010Inngår i: ACTA OPHTHALMOLOGICA, ISSN 1755-375X, Vol. 88, nr 4, s. 420-425Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Purpose: To investigate the effects on near visual acuity, reading speed, central visual field and related quality of life of ranibizumab treatment of wet age-related macular degeneration (AMD). Methods: The study was a prospective, non-comparative consecutive case series, followed for 3 months and investigator-driven. Thirty eyes of 30 patients with wet AMD were included, mean age 75 years (range 69-95 years). In addition to a full ophthalmological examination - including best-corrected visual acuity (BCVA; Early Treatment Diabetic Research Study chart), fundus biomicroscopy, fundus photography, fluorescein angiography, indocyanine green angiography (occult cases) and ocular coherence tomography - near visual acuity, reading speed, central visual field and quality of life for related activities were also investigated at baseline and at 3 months after ranibizumab treatment. Results: Mean BCVA increased from 62 +/- 11 to 66 +/- 14 letters at 3 months (7%; p = 0.018). Near vision improved from 9 +/- 5 to 6 +/- 3 points (33%; p = 0.0006) and reading speed increased from 59 +/- 40 to 85 +/- 50 words/min (44%; p andlt; 0.0001). The mean deviation from normal of the visual field improved from -9 +/- 7 to -6 +/- 5 dB (33%; p andlt; 0.0001). Quality of life improved for distance activities from 54 +/- 28 to 63 +/- 28 points (17%; p andlt; 0.0001) but significantly (p = 0.024) more for near activities, from 49 +/- 26 to 63 +/- 26 points (29%; p andlt; 0.0001). Reading newspaper text in the group in which the better eye was treated showed the highest increase in quality of life score of all: 116%. Conclusion: The increase in BCVA after ranibizumab treatment is well established. The present study also showed significant improvements in other important visual qualities, such as near visual acuity, reading speed, central visual field and several activities influencing quality of life. The improvement was greater for near activities than for distance activities. Therefore, the beneficial effects of ranibizumab treatment shown here are more extensive than those reported previously.

  • 11.
    Heintz, Emelie
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Utvärdering och hälsoekonomi. Linköpings universitet, Hälsouniversitetet.
    Bourghardt Peebo, Beatrice
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Wiréhn, Ann-Britt
    Linköpings universitet, Institutionen för medicin och hälsa, Hälsa och samhälle. Linköpings universitet, Hälsouniversitetet.
    Rosenqvist, Ulf
    Östergötlands Läns Landsting, Närsjukvården i västra Östergötland, Medicinska specialistkliniken .
    Levin, Lars-Åke
    Linköpings universitet, Institutionen för medicin och hälsa, Utvärdering och hälsoekonomi. Linköpings universitet, Hälsouniversitetet.
    Health-related quality of life profiles of patients with diabetic retinopathyManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Purpose: There are various instruments for estimating health-related quality of life (HRQoL) in patients with diabetic retinopathy (DR). However, if the results are to be compared with those for other diseases, it is essential to use measures that are applicable to all disease areas. The aim of this study was to explore the HRQoL profiles of patients with DR using two generic multi-attribute instruments, the Health State Utilities Index Mark 3 (HUI-3) and the EQ-5D questionnaire, and to investigate these questionnaires’ sensitivity to differences in HRQoL due to DR.

    Methods: The study population comprised 166 Swedish diabetes patients diagnosed with DR at different severities. Patients were interviewed over the telephone using HUI-3 and EQ-5D. The vision-specific National Eye Institute Visual Functioning Questionnaire 25 (NEI VFQ-25) was also included, to give an empirical framework for the results of the generic instruments. Linear and logistic regression models were used to adjust for possible confounders.

    Results: Patients with vision impairment (VI) reported lower scores in Vision, Ambulation, and Pain in HUI-3 and more problems with Usual activities and Anxiety/depression in EQ-5D. However, even though NEI VFQ-25 showed a negative association between DR severity and Mental health and Near activities, neither EQ-5D nor HUI-3 identified a negative relationship between DR severity and HRQoL.

    Conclusion: The generic instruments show lowered HRQoL for patients with VI in various dimensions but were not sensitive to decrements related to the diagnosis of DR alone. The questionnaire of HUI-3 was more sensitive than EQ-5D to differences in HRQoL due to DR-related VI.

  • 12.
    Heintz, Emelie
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Utvärdering och hälsoekonomi. Linköpings universitet, Hälsouniversitetet.
    Wiréhn, Ann-Britt
    Linköpings universitet, Institutionen för medicin och hälsa, Hälsa och samhälle. Linköpings universitet, Hälsouniversitetet.
    Bourghardt Peebo, Beatrice
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Rosenqvist, Ulf
    Östergötlands Läns Landsting, Närsjukvården i västra Östergötland, Medicinska specialistkliniken .
    Levin, Lars-Åke
    Linköpings universitet, Institutionen för medicin och hälsa, Utvärdering och hälsoekonomi. Linköpings universitet, Hälsouniversitetet.
    QALY weights for diabetic retinopathy: a comparison of health state valuations with HUI-3, EQ-5D, EQ-VAS, and TTO.2012Inngår i: Value in Health, ISSN 1098-3015, E-ISSN 1524-4733, Vol. 15, nr 3, s. 475-484Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Objective: To estimate quality-adjusted life-year weights for patients with diabetic retinopathy by using various methods and to investigate the empirical validity of the different measures.

    Methods: The study population comprised 152 patients with diabetes in Östergötland County, Sweden. Participants were interviewed by telephone by using the time trade-off (TTO) method and a visual analogue scale (EQ-VAS) (direct valuations) as well as the EuroQol five-dimensional questionnaire (EQ-5D) and the health utilities index mark 3 (HUI-3) (indirect valuations). The quality-adjusted life-year weights were adjusted for potential confounders by using analysis of covariance. The empirical validity of the measures was examined by testing their ability to detect hypothetical differences between severity levels of diabetic retinopathy and by investigating the correlation between the measures and the 25-item National Eye Institute Visual Function Questionnaire (NEI VFQ-25).

    Results: All measures detected significant differences in scores between patient groups classified according to visual impairment in the better eye (analysis of covariance, P < 0.05), but only HUI-3 and EQ-VAS detected significant differences between patient groups classified according to visual impairment or pathological progression in the worse eye. HUI-3 recorded a difference of 0.43 in values between normal vision and blindness in the better eye, which was more than twice the differences captured by the other measures (0.15–0.20). In addition, HUI-3 showed the highest correlation with NEI VFQ-25 (r = 0.54; P < 0.001).

    Conclusions: In cost-utility analyses, the choice of quality-adjusted life-year measure may affect whether an intervention is considered cost-effective. Furthermore, if decisions are to be based on values from the general public, HUI-3 can be recommended for cost-utility analyses of interventions directed at diabetic retinopathy.

  • 13.
    Heintz, Emelie
    et al.
    Linköpings universitet, Institutionen för medicin och hälsa, Utvärdering och hälsoekonomi. Linköpings universitet, Hälsouniversitetet.
    Wiréhn, Ann-Britt
    Linköpings universitet, Institutionen för medicin och hälsa, Hälsa och samhälle. Linköpings universitet, Hälsouniversitetet.
    Peebo Bourghardt, Beatrice
    Östergötlands Läns Landsting, Rekonstruktionscentrum, Ögonkliniken US/LiM.
    Rosenqvist, U
    Motala Hospital, Sweden.
    Levin, Lars-Åke
    Linköpings universitet, Institutionen för medicin och hälsa, Utvärdering och hälsoekonomi. Linköpings universitet, Hälsouniversitetet.
    Prevalence and healthcare costs of diabetic retinopathy: a population-based register study in Sweden2010Inngår i: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 53, nr 10, s. 2147-2154Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Aims/hypothesis

    The aim of the present study was to estimate the prevalence and healthcare costs of diabetic retinopathy (DR).

    Methods

    This population-based study included all residents (n = 251,386) in the catchment area of the eye clinic of Linköping University Hospital, Sweden. Among patients with diabetes (n  = 12,026), those with and without DR were identified through register data from both the Care Data Warehouse in Östergötland, an administrative healthcare register, and the Swedish National Diabetes Register. Healthcare cost data were elicited by record linkage of these two registers to data for the year 2008 in the Cost Per Patient Database developed by Östergötland County Council.

    Results

    The prevalence of any DR was 41.8% (95% CI 38.9–44.6) for patients with type 1 diabetes and 27.9% (27.1–28.7) for patients with type 2 diabetes. Sight-threatening DR was present in 12.1% (10.2–14.0) and 5.0% (4.6–5.4) of the type 1 and type 2 diabetes populations respectively. The annual average healthcare cost of any DR was €72 (€53–91). Stratified into background retinopathy, proliferative DR, maculopathy, and the last two conditions combined, the costs were €26 (€10–42), €257 (€155–359), €216 (€113–318) and €433 (€232–635) respectively. The annual cost for DR was €106 000 per 100,000 inhabitants.

    Conclusions

    This study presents new information on the prevalence and costs of DR. Approximately one-third of patients with diabetes have some form of DR. Average healthcare costs increase considerably with the severity of DR, which suggests that preventing progression of DR may lower healthcare costs.

  • 14.
    Lagali, Neil
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Bourghardt Peebo, Beatrice
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Germundsson, Johan
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Edén, Ulla
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    Danyali, Reza
    Linköpings universitet, Hälsouniversitetet.
    Rinaldo, Marcus
    Linköpings universitet, Hälsouniversitetet.
    Fagerholm, Per
    Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Laser-Scanning in vivo Confocal Microscopy of the Cornea: Imaging and Analysis Methods for Preclinical and Clinical Applications2013Inngår i: Confocal Laser Microscopy: Principles and Applications in Medicine, Biology, and the Food Sciences, INTECH, 2013Kapittel i bok, del av antologi (Annet vitenskapelig)
  • 15.
    Mirabelli, Pierfrancesco
    et al.
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neurovetenskap. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Bourghardt Peebo, Beatrice
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neurovetenskap. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Xeroudaki, Maria
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neurovetenskap. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Koulikovska, Marina
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neurovetenskap. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Lagali, Neil
    Linköpings universitet, Institutionen för klinisk och experimentell medicin, Avdelningen för neurovetenskap. Linköpings universitet, Hälsouniversitetet. Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM.
    Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model2014Inngår i: Experimental Eye Research, ISSN 0014-4835, E-ISSN 1096-0007, Vol. 125, s. 118-127Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Inflammatory angiogenesis is the pathogenic mechanism of various sight-threatening eye diseases, among them corneal neovascularization. Current treatment options include steroids which have undesirable side effects, or anti-VEGF which has only limited efficacy. In an inflammatory environment, however, angiogenesis can be stimulated by numerous factors not directly targeted by anti-VEGF therapy. The aim of this study was to induce corneal inflammation leading to angiogenesis, and investigate the early, differential effects of steroid and anti-VEGF therapy at the cellular, tissue, and gene expression levels. Fifty-two Wistar rats received a single intrastromal corneal suture to induce a controlled inflammatory angiogenic response. Rats were subsequently treated with dexamethasone, rat specific anti-VEGF, or goat IgG (control), topically 4 times daily for 7 days. In vivo confocal microscopy of the cornea was performed longitudinally from 5 h up to 7 d to investigate morphology at the cellular and tissue-level. In vivo photographic vessel analysis and immunohistochemistry were also performed. RT-PCR for VEGF-A, FGF-2, IL-6, TNF-alpha, CXCL2, CCL2, CCL3 and DLL4 was performed at 24 h, and for VEGF-A, IL-6, TNF-alpha, FGF-2, CXCL2, CCL2, and CCL3 at 7 days. Early infiltration of CD11b + myeloid cells into the cornea at 5 h post-suture was delayed by both treatments relative to controls; however neither treatment was able to suppress accumulation of myeloid cells at day 2 or 7. Limbal vessel dilation was inhibited at 5 h by both treatments, but only dexamethasone showed sustained effect until day 2. Early macrophage recruitment was also suppressed by dexamethasone (but not by anti-VEGF) until day 2. Dexamethasone furthermore suppressed corneal neovascularization at day 7 by over 90%, whereas suppression by anti-VEGF was 14%. Despite differential suppression of vessel dilation, macrophage recruitment, and vascular invasion, anti-VEGF and dexamethasone both down-regulated VEGF-A and IL-6 expression at 24 h with sustained effect to 7 d. They also both down regulated FGF-2 and TNF-alpha at 24 h and CCL2 at 7 d. In conclusion, anti-angiogenic treatments influence early, pre-angiogenic tissue activity such as limbal vessel dilation, inflammatory cell infiltration of the stroma, and macrophage recruitment. Importantly, the differential effects of steroids and anti-VEGF treatment in suppressing neovascular growth could not be attributed to differential inhibition of several major angiogenic and inflammatory factors in the early pre-sprouting phase, including IL-6, VEGF-A, FGF-2, TNF-alpha, CCL2, CCL3, CXCL2, or DLL4.

  • 16.
    Wiklund, Anna
    et al.
    Karolinska Institutet, Institutionen för klinisk neurovetenskap,.
    Bourghardt Peebo, Beatrice
    Östergötlands Läns Landsting, Sinnescentrum, Ögonkliniken US/LiM. Linköpings universitet, Institutionen för klinisk och experimentell medicin. Linköpings universitet, Hälsouniversitetet.
    ACUTE EXUDATIVE POLYMORPHOUS VITELLIFORM MACULOPATHY IN A YOUNG WOMAN: PRESYMPTOMATIC FINDINGS AND 21-MONTH FOLLOW-UP2013Inngår i: RETINAL Cases & Brief Reports, ISSN 1935-1089, Vol. 7, nr 2, s. 123-127Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Purpose: To describe ocular findings before and after the diagnosis of acute exudative polymorphous vitelliform maculopathy, in an otherwise healthy 28-year-old woman.

    Methods: Case report with 21-months of follow-up. Fundus photography, optical coherence tomography, fluorescein angiography, indocyanine green angiography, and autofluorescence were used for imaging the retina. To examine retinal function, full-field electroretinogram, multifocal electroretinogram, electrooculography, and dark adaptometry were performed. Genetic analysis for mutations associated with Best disease was done.

    Results: In the asymptomatic patient before diagnosis, white-yellow, drusen-like, subretinal depositions were found in both eyes. A few months later, the patient developed bilateral visual disturbances. Retinal examination at the acute phase revealed a characteristic pattern of multifocal white-yellow subretinal lesions in both posterior poles, imaged by ophthalmoscopy, fluorescein angiography, indocyanine green angiography, and optical coherence tomography. Additionally, electrooculography and dark adaptometry were abnormal. Full-field electroretinogram was normal, but multifocal electroretinogram revealed central depression of peak amplitudes. During the 21-month follow-up without any treatment, visual acuity recovered, electrooculography and dark adaptometry normalized, and the patient experienced one episode of relapse. Genetic studies excluded mutations in the bestrophin gene (BEST1).

    Conclusion: Acute exudative polymorphous vitelliform maculopathy is still a condition of unknown origin, primarily affecting the pigment epithelium. Earlier reports have discussed whether the condition is inherited or acquired. In this report, the presymptomatic retinal findings in acute exudative polymorphous vitelliform maculopathy are described for the first time, indicating that a condition may be associated with primarily affected retinal pigment epithelium.

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