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1.
J Glaucoma ; 29(12): 1101-1105, 2020 12.
Article in English | MEDLINE | ID: mdl-32890107

ABSTRACT

PRéCIS:: Regardless of the blood-filling patterns in Schlemm canal (SC) before the trabecular meshwork (TM) ablation, the trabectome surgery, combined with phacoemulsification, is effective for mild to moderate primary open-angle glaucoma patients. PURPOSE: The purpose of this study was to evaluate the association between trabectome surgery outcomes and the blood filling patterns in SC before TM ablation. MATERIALS AND METHODS: This retrospective cohort study included 105 eyes of 84 Japanese primary open-angle glaucoma patients who had undergone trabectome surgery in combination with cataract surgery. Provocative gonioscopy was performed before TM ablation to classify the blood filling patterns in SC into 3 groups: no filling (group 1); patchy/irregular filling (group 2); and complete filling (group 3). The subjects were divided into 3 groups according to the blood filling patterns and the trabectome surgery outcomes were compared, including intraocular pressure (IOP), the percentage reduction in IOP, surgical success rate, and the number of glaucoma medications. Success was defined by IOP ≤15 mm Hg and a >20% reduction in IOP with/without glaucoma medication, and without additional glaucoma surgery after trabectome surgery combined with cataract surgery. RESULTS: Twenty-four eyes were assigned to group 1, 48 to group 2, and 33 to group 3. Between-group analyses showed no significant intergroup differences in age (P=0.213), preoperative mean deviation (P=0.505), preoperative and postoperative IOP (P=0.941 and 0.458, respectively), preoperative and postoperative number of glaucoma medications (P=0.805 and 0.077, respectively), percentage IOP reduction (P=0.256), and success rates (P=0.540). CONCLUSION: Trabectome surgery is effective for mild to moderate primary open-angle glaucoma patients, independent of the blood-filling patterns in SC before the TM ablation.


Subject(s)
Glaucoma, Open-Angle/surgery , Limbus Corneae/blood supply , Sclera/blood supply , Trabecular Meshwork/surgery , Trabeculectomy , Aged , Aged, 80 and over , Female , Follow-Up Studies , Glaucoma, Open-Angle/diagnosis , Glaucoma, Open-Angle/physiopathology , Gonioscopy , Humans , Intraocular Pressure/physiology , Male , Middle Aged , Phacoemulsification , Retrospective Studies , Tonometry, Ocular
2.
Exp Eye Res ; 201: 108278, 2020 12.
Article in English | MEDLINE | ID: mdl-32997957

ABSTRACT

PURPOSE: To describe the location and morphometric characteristics of the human limbal lymphatic vasculature and its relation to the marginal corneal vascular arcades (MCA). METHODS: Ex vivo confocal microscopic (CM) imaging and immunofluorescence double staining for CD-31 and D2-40 of histological en-face sections using 12 preserved human cadaveric corneoscleral discs were performed, followed by a semi-automated morphometric analysis of the two-dimensional vascular network architecture. RESULTS: Ex vivo CM confirmed the presence of 2 distinct vascular networks. The haematic limbal vascular complex (HLVC) extended further into the cornea, forming typical MCAs. The lymphatic limbal vascular complex (LLVC) was peripheral from the termination of Bowman's layer and was also found to be peripheral to and deeper than the HLVC. LLVC and HLVC were significantly different with respect to vessel diameter, segment length and wall thickness. CONCLUSION: The lymphatic vasculature of the human corneoscleral limbal region displays specific morphometric features that allow its differentiation from haematic vessels using CM.


Subject(s)
Limbus Corneae/blood supply , Lymphatic Vessels/diagnostic imaging , Microscopy, Confocal/methods , Aged , Epithelium, Corneal/cytology , Female , Humans , Male
3.
Invest Ophthalmol Vis Sci ; 61(2): 23, 2020 02 07.
Article in English | MEDLINE | ID: mdl-32068793

ABSTRACT

Purpose: To validate the ability of visible-light optical coherence tomography (vis-OCT) in imaging the full Schlemm's canal (SC) and its surrounding limbal vascular network in mice in vivo through a compound circumlimbal scan. Methods: We developed an anterior segment vis-OCT system and a compound circumlimbal scanning method, which montages eight rotated raster scans. We calibrated the circumlimbal scan geometry using a three-dimensional printed phantom eyeball before imaging wild-type C57BL/6J mice. We measured SC size by segmenting SC cross sections from vis-OCT B-scan images and imaged the limbal microvascular network using vis-OCT angiography (vis-OCTA). To introduce changes in SC size, we used a manometer to adjust the intraocular pressure (IOP) to different levels. To create additional optical scattering contrast to enhance SC imaging, we surgically increased the episcleral venous pressure (EVP) and caused blood reflux into SC. Results: Using the compound circumlimbal scan, our anterior segment vis-OCT noninvasively imaged the full SC and limbal microvascular network in mouse for the first time. We observed an average 123% increase in SC volume when we decreased the IOP by 10 mm Hg from the baseline IOP of 7 to 10 mm Hg and an average 72% decrease in SC volume when the IOP level was elevated by 10 mm Hg from the baseline IOP. We also observed location-dependent SC size responses to IOP changes. Blood reflux caused by increased EVP enabled vis-OCTA to directly visualize SC, which matched well with the segmented SC. Conclusions: Vis-OCT and vis-OCTA can accurately image the entire SC and limbal microvascular network in vivo using the compound circumlimbal scan. Vis-OCT is also able to quantitatively measure SC responses to changing IOP levels.


Subject(s)
Limbus Corneae , Sclera , Tomography, Optical Coherence/methods , Animals , Limbus Corneae/blood supply , Limbus Corneae/diagnostic imaging , Mice , Mice, Inbred C57BL , Sclera/blood supply , Sclera/diagnostic imaging
5.
Sci Rep ; 9(1): 12331, 2019 08 23.
Article in English | MEDLINE | ID: mdl-31444394

ABSTRACT

The role of the corneal epithelium and limbus in corneal avascularity and pathological neovascularization (NV) is not well understood. To investigate the contributions of the corneal and limbal epithelia in angiogenic and lymphangiogenic privilege, we designed five injury models involving debridement of different portions of the cornea and limbus and applied them to the dual-fluorescence reporter Prox1-GFP/Flt1-DsRed mouse, which permits in vivo imaging of blood and lymphatic vessels via fluorescence microscopy. Debridement of the whole cornea resulted in significant hemangiogenesis (HA) and lymphangiogenesis (LA), while that of the whole limbus yielded minimal corneal HA or LA. Following hemilimbal plus whole corneal debridement, corneal NV occurred only through the non-injured aspect of the limbus. Overall, these results suggest that the integrity of the corneal epithelium is important for (lymph)angiogenic privilege, whereas the limbus does not act as a physical or physiologic barrier to invading vessels. In CDh5-CreERT2VEGFR2lox/PGFD mice, conditional deletion of vascular endothelial growth factor receptor 2 in vascular endothelial cells abolished injury-induced HA and LA, demonstrating the utility of this transgenic mouse line for identifying important factors in the process of neovascularization.


Subject(s)
Corneal Injuries/pathology , Fluorescent Dyes/metabolism , Limbus Corneae/blood supply , Limbus Corneae/pathology , Lymphangiogenesis , Neovascularization, Physiologic , Animals , Debridement , Disease Models, Animal , Epithelium, Corneal/pathology , Mice, Transgenic
6.
Am J Ophthalmol ; 208: 313-322, 2019 12.
Article in English | MEDLINE | ID: mdl-31102577

ABSTRACT

PURPOSE: To investigate conjunctival and intrascleral vasculature in glaucoma eyes using anterior segment (AS)-optical coherence tomography angiography (OCTA) and assess the factors contributing to the vessel density in AS-OCTA images. DESIGN: Prospective, cross-sectional study. METHODS: Thirty-four patients with primary open-angle glaucoma and 20 healthy subjects were included. A swept-source OCT system was used to obtain the AS-OCTA images of the corneoscleral limbus at the nasal and temporal quadrants. Vessel densities were measured in the superficial (from the conjunctival epithelium to a depth of 200 µm) and deep (from a depth of 200 to 1000 µm) layers. The vessel density was compared between healthy and glaucoma eyes, and the associations of the vessel density with possible confounding factors were analyzed using univariable and multivariable analyses. RESULTS: The vessel density was not significantly different between healthy eyes and eyes with glaucoma. There was a significant association of superficial vessel density with the use of a prostaglandin analog (P = .007) and with nasal location (P = .016) in eyes with glaucoma. Deep vessel density was significantly smaller with advancing age (P = .029) in healthy eyes and greater with higher intraocular pressure (P = .021) in eyes with treated glaucoma. CONCLUSIONS: AS-OCTA images may be useful for the objective assessment of conjunctival hyperemia and helpful for understanding the pathophysiology of post-trabecular aqueous humor outflow.


Subject(s)
Conjunctiva/blood supply , Glaucoma, Open-Angle/physiopathology , Sclera/blood supply , Adult , Aged , Aged, 80 and over , Anterior Eye Segment/diagnostic imaging , Conjunctiva/diagnostic imaging , Cross-Sectional Studies , Female , Fluorescein Angiography , Glaucoma, Open-Angle/diagnosis , Gonioscopy , Humans , Intraocular Pressure/physiology , Limbus Corneae/blood supply , Limbus Corneae/diagnostic imaging , Male , Middle Aged , Prospective Studies , Sclera/diagnostic imaging , Slit Lamp Microscopy , Tomography, Optical Coherence , Tonometry, Ocular , Visual Field Tests , Visual Fields/physiology
7.
Indian J Ophthalmol ; 67(1): 32-36, 2019 Jan.
Article in English | MEDLINE | ID: mdl-30574887

ABSTRACT

PURPOSE: Limbal ischemia is an important prognostic factor in the management of ocular burns. In this study, we evaluated the reliability of clinically assessing limbal ischemia among ophthalmic professionals. METHODS: This study included 111 ophthalmic professionals who were shown 12 diffuse illumination color slit-lamp photographs of eyes with recent chemical injuries. Respondents were asked whether the photos were assessable and if yes, then to indicate the presence, location, and grade of limbal ischemia in each case. The responses were collected using a standard data collection sheet and the inter-observer agreement was calculated. RESULTS: All participants responded to every question. Of the 1,332 responses, images were deemed assessable in 1,222 (91.7%) instances. The overall agreement (Fleiss' kappa) for the presence of limbal ischemia and severity of limbal ischemia was 0.106 and 0.139, respectively (P < 0.012). Among the four groups of observers, practicing cornea specialists displayed significantly (P < 0.003) higher kappa values (0.201-0.203) when compared to residents (0.131-0.185), fellows (0.086-0.127), and optometrists (0.077-0.102). All indicated a poor level of inter-rater consistency. CONCLUSION: The results indicate that clinical assessment of limbal ischemia is highly subjective and there is lack of reliability even among cornea specialists who regularly manage patients with ocular burns. A non-invasive, standardized, objective, accurate, and reliable modality for ocular surface angiography is desperately needed for proper assessment and prognostication of ocular burns.


Subject(s)
Burns, Chemical/surgery , Disease Management , Eye Burns/surgery , Ischemia/etiology , Limbus Corneae/blood supply , Stem Cell Transplantation/methods , Adult , Burns, Chemical/complications , Burns, Chemical/diagnosis , Eye Burns/complications , Eye Burns/diagnosis , Female , Humans , Ischemia/diagnosis , Ischemia/surgery , Limbus Corneae/diagnostic imaging , Limbus Corneae/injuries , Male , ROC Curve , Slit Lamp Microscopy , Visual Acuity
8.
Invest Ophthalmol Vis Sci ; 58(10): 3879-3886, 2017 08 01.
Article in English | MEDLINE | ID: mdl-28763561

ABSTRACT

Purpose: This study was designed to develop and characterize a laser-induced model of acute intraocular hypertension that permits the study of the anterior segment of the eye. Methods: CD1 mice aged 5 and 8 weeks were examined for elevation of IOP induced by laser photocoagulation. We compared between occlusion of episcleral veins alone and when combined with 270° limbal vessel occlusion. Anterior chamber angle, corneal thickness, and retinal nerve fiber layer (RNFL) thickness were evaluated by anterior- and posterior-segment optical coherence tomography (OCT). Additionally, at day 7 post-procedure, the anterior segment was evaluated for inflammatory cellular presentation by histologic analysis and OCT, and limbal vessels and whole-mount retina were immunostained for CD31 and Brn3a, respectively. Brn3a-positive retinal ganglion cells (RGCs) were quantified with ImageJ software. Results: After single or combined laser treatment in mice aged 5 or 8 weeks, IOP was significantly elevated for 5 to 6 days before returning to the baseline by day 7 post-procedure. Anterior segment assessment indicated less synechiae in the anterior chamber angle and better preserved limbal vessels with single versus combined laser treatment. Corneal thickness was significantly increased after single or combined treatment. No inflammatory cells were detected in the anterior chamber. The thickness of the RNFL and the density of RGCs were both significantly reduced after single or combined treatment. Conclusions: Laser photocoagulation of episcleral veins alone in CD1 mice aged 5 to 8 weeks may be used to induce ocular hypertension resulting in RNFL thinning and ganglion cell loss. This model permits the study of the anterior as well as the posterior segment of the eye.


Subject(s)
Intraocular Pressure/radiation effects , Light Coagulation/adverse effects , Ocular Hypertension/pathology , Ocular Hypertension/physiopathology , Retinal Vein Occlusion/pathology , Acute Disease , Animals , Disease Models, Animal , Limbus Corneae/blood supply , Limbus Corneae/pathology , Optic Disk/pathology , Retina/pathology , Retinal Ganglion Cells/pathology , Sclera/blood supply , Tomography, Optical Coherence , Veins
9.
Sci Rep ; 7(1): 6377, 2017 07 25.
Article in English | MEDLINE | ID: mdl-28743889

ABSTRACT

Limbal stromal cells (LSCs) from the human ocular surface display mesenchymal stromal cell characteristics in vitro. In this study, we isolated cells from the porcine limbal stroma (pLSCs), characterised them, and evaluated their ability to support angiogenesis and the culture of porcine limbal epithelial stem cells (pLESCs). The isolated cells adhered to plastic and grew in monolayers in vitro using serum-supplemented or serum-free medium. The pLSCs demonstrated expression of CD29, and cross-reactivity with anti-human CD45, CD90, CD105, CD146, and HLA-ABC. However, expression of CD105, CD146 and HLA-ABC reduced when cultured in serum-free medium. PLSCs did not undergo adipogenic or osteogenic differentiation, but differentiated towards the chondrogenic lineage. Isolated cells were also co-cultured with human umbilical vein endothelial cells (HUVECs) in star-shaped Poly(ethylene glycol) (starPEG)-heparin hydrogels to assess their pericyte capacity which supported angiogenesis networks of HUVECs. PLSCs supported the three dimensional HUVEC network for 7 days. The isolated cells were further growth-arrested and evaluated as feeder cells for pLESC expansion on silk fibroin membranes, as a potential carrier material for transplantation. PLSCs supported the growth of pLESCs comparably to murine 3T3 cells. In conclusion, although pLSCs were not completely comparable to their human counterpart, they display several mesenchymal-like characteristics in vitro.


Subject(s)
Cell Culture Techniques/methods , Integrin beta1/metabolism , Limbus Corneae/cytology , Mesenchymal Stem Cells/cytology , Animals , Cell Adhesion , Cell Differentiation , Cell Proliferation , Cells, Cultured , Coculture Techniques , Feeder Cells/cytology , Human Umbilical Vein Endothelial Cells , Humans , Limbus Corneae/blood supply , Limbus Corneae/metabolism , Mesenchymal Stem Cells/metabolism , Mice , NIH 3T3 Cells , Neovascularization, Physiologic , Stromal Cells , Swine
10.
Exp Eye Res ; 146: 293-303, 2016 05.
Article in English | MEDLINE | ID: mdl-27068508

ABSTRACT

Mutations in the FOXC1/Foxc1 gene in humans and mice and Bmp4 in mice are associated with congenital anterior segment dysgenesis (ASD) and the development of the aqueous outflow structures throughout the limbus. The aim of this study was to advance our understanding of anterior segment abnormalities in mouse models of ASD using a 3-D imaging approach. Holistic imaging information combined with quantitative measurements were carried out on PECAM-1 stained individual components of the aqueous outflow vessels and corneal vasculature of Foxc1(+/-) on the C57BL/6Jx129 and ICR backgrounds, Bmp4(+/-) ICR mice, and wildtype mice from each background. In both wildtype and heterozygotes, singular, bifurcated and plexus forms of Schlemm's canal were noted. Of note, missing portions of the canal were seen in the heterozygous groups but not in wildtype animals. In general, we found the number of collector channels to be reduced in both heterozygotes. Lastly, we found a significant increase in the complexity of the corneal arcades and their penetration into the cornea in heterozygotes as compared with wild types. In conclusion, our 3-D imaging studies have revealed a more complex arrangement of both the aqueous vessels and corneal arcades in Foxc1(+/-) and Bmp4(+/-) heterozygotes, and further advance our understanding of how such abnormalities could impact on IOP and the aetiology of glaucoma.


Subject(s)
Anterior Eye Segment/anatomy & histology , Aqueous Humor/metabolism , Bone Morphogenetic Protein 4/physiology , Forkhead Transcription Factors/physiology , Limbus Corneae/blood supply , Analysis of Variance , Animals , Anterior Eye Segment/blood supply , Blood Vessels/anatomy & histology , Bone Morphogenetic Protein 4/deficiency , Choroid/blood supply , Forkhead Transcription Factors/deficiency , Heterozygote , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Mice, Transgenic , Microscopy, Confocal
11.
Br J Ophthalmol ; 100(3): 315-22, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26163540

ABSTRACT

BACKGROUND/AIMS: To clinically characterise corneal neovascularisation (CVas) with a view to elaborate clinical presentations and to standardise descriptors for clinical evaluation and research. METHODS: Corneas of 165 patients with CVas due to a variety of corneal pathologies were observed clinically with the slit lamp biomicroscope and photography at different time points over the course of their disease. Parameters assessed included location, depth, length, branching pattern, colour, lipid leakage, nature of blood flow and presence of haemorrhage. A clinical grading of CVas was applied. RESULTS: CVas can arise from the limbus, conjunctiva and iris. CVas preferentially travels along planes created by corneal incisions, suture tracks and lamellar keratoplasty, both deep lamellar and endothelial keratoplasty. CVas also principally follows the depth of pathology. CVas can be classified into active young, active old, mature, partially regressed and regressed. Herpetic infection was the most common cause of lipid keratopathy. Acanthamoeba keratitis induced the least amount of vascularisation. A simple and efficient clinical grading system for ascertaining the severity of CVas was developed and validated. CONCLUSIONS: The various clinical characteristics of CVas described in this study can be used to standardise the nomenclature and description of CVas and clinically grade its severity. As modern, effective methods of treating CVas are being introduced, it is important that there is uniformity in the descriptors used to establish baseline values and evaluate outcomes of treatment. The parameters established in this study can help in this regard.


Subject(s)
Conjunctiva/blood supply , Corneal Neovascularization/diagnosis , Iris/blood supply , Limbus Corneae/blood supply , Adult , Conjunctiva/pathology , Corneal Neovascularization/classification , Corneal Neovascularization/etiology , Corneal Neovascularization/physiopathology , Female , Humans , Iris/pathology , Limbus Corneae/pathology , Male , Middle Aged , Photography , Prospective Studies , Retrospective Studies , Slit Lamp
12.
Stem Cells Transl Med ; 4(9): 1052-63, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26185258

ABSTRACT

UNLABELLED: Stem cell-based therapy has become an attractive and promising approach for the treatment of severe injuries or thus-far incurable diseases. However, the use of stem cells is often limited by a shortage of available tissue-specific stem cells; therefore, other sources of stem cells are being investigated and tested. In this respect, mesenchymal stromal/stem cells (MSCs) have proven to be a promising stem cell type. In the present study, we prepared MSCs from bone marrow (BM-MSCs) or adipose tissue (Ad-MSCs) as well as limbal epithelial stem cells (LSCs), and their growth, differentiation, and secretory properties were compared. The cells were grown on nanofiber scaffolds and transferred onto the alkali-injured eye in a rabbit model, and their therapeutic potential was characterized. We found that BM-MSCs and tissue-specific LSCs had similar therapeutic effects. Clinical characterization of the healing process, as well as the evaluation of corneal thickness, re-epithelialization, neovascularization, and the suppression of a local inflammatory reaction, were comparable in the BM-MSC- and LSC-treated eyes, but results were significantly better than in injured, untreated eyes or in eyes treated with a nanofiber scaffold alone or with a nanofiber scaffold seeded with Ad-MSCs. Taken together, the results show that BM-MSCs' therapeutic effect on healing of injured corneal surface is comparable to that of tissue-specific LSCs. We suggest that BM-MSCs can be used for ocular surface regeneration in cases when autologous LSCs are absent or difficult to obtain. SIGNIFICANCE: Damage of ocular surface represents one of the most common causes of impaired vision or even blindness. Cell therapy, based on transplantation of stem cells, is an optimal treatment. However, if limbal stem cells (LSCs) are not available, other sources of stem cells are tested. Mesenchymal stem cells (MSCs) are a convenient type of cell for stem cell therapy. The therapeutic potential of LSCs and MSCs was compared in an experimental model of corneal injury, and healing was observed following chemical injury. MSCs and tissue-specific LSCs had similar therapeutic effects. The results suggest that bone marrow-derived MSCs can be used for ocular surface regeneration in cases when autologous LSCs are absent or difficult to obtain.


Subject(s)
Burns, Chemical/therapy , Cell- and Tissue-Based Therapy/methods , Epithelial Cells/physiology , Epithelium, Corneal/injuries , Limbus Corneae/injuries , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/physiology , Adipocytes/cytology , Adipocytes/physiology , Adipose Tissue/cytology , Adipose Tissue/physiology , Animals , Biomarkers/metabolism , Bone Marrow Cells/cytology , Bone Marrow Cells/physiology , Burns, Chemical/pathology , Cell Differentiation , Cell Proliferation , Epithelial Cells/cytology , Epithelial Cells/transplantation , Epithelium, Corneal/blood supply , Female , Gene Expression , Limbus Corneae/blood supply , Mesenchymal Stem Cells/cytology , Neovascularization, Physiologic , Primary Cell Culture , Rabbits , Re-Epithelialization/physiology , Tissue Scaffolds
13.
Cell Tissue Res ; 359(2): 547-563, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25398719

ABSTRACT

Knowledge of the microenvironment (niche) of stem cells is helpful for stem-cell-based regenerative medicine. In the eye, limbal epithelial stem cells (corneal epithelial stem cells) provide the self-renewal capacity of the corneal epithelium and are essential for maintaining corneal transparency and vision. Limbal epithelial stem cell deficiency results in significant visual deterioration. Successful treatment of this type of blinding disease requires studies of the limbal epithelial stem cells and their microenvironment. We investigate the function of the limbal microvascular net and the limbal stroma in the maintenace of the limbal epithelial stem cell niche in vivo and examine the regulation of limbal epithelial stem cell survival, proliferation and differentiation in vivo. We assess the temporal and spatial changes in the expression patterns of the following markers during a six-month follow-up of various rabbit limbal autograft transplantation models: vascular endothelial cell marker CD31, corneal epithelium differentiation marker K3, limbal epithelial stem-cell-associated markers P63 and ABCG2 and proliferating cell nuclear marker Ki67. Our results suggest that limbal epithelial stem cells cannot maintain their stemness or proliferation without the support of the limbal microvascular net microenvironment. Thus, both the limbal microvascular net and the limbal stroma play important roles as components of the limbal epithelial stem cell niche maintaining limbal epithelial stem cell survival and proliferation and the avoidance of differentiation. The limbal stroma constitutes the structural basis of the limbal epithelial stem cell niche and the limbal microvascular net is a requirement for this niche. These new insights should aid the eventual construction of tissue-engineered cornea for corneal blind patients in the future.


Subject(s)
Corneal Stroma/cytology , Epithelial Cells/cytology , Limbus Corneae/blood supply , Limbus Corneae/cytology , Microvessels/metabolism , Stem Cells/cytology , ATP-Binding Cassette Transporters/metabolism , Animals , Autografts , Biomarkers/metabolism , Cell Differentiation , Cell Nucleus/metabolism , Cell Proliferation , Fluorescent Antibody Technique , Ki-67 Antigen/metabolism , Models, Animal , Rabbits , Slit Lamp , Staining and Labeling , Time Factors , Transplantation, Autologous
14.
Exp Eye Res ; 131: 20-8, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25499210

ABSTRACT

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with foetal bovine serum (10% v/v), D-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 µg/mL), hydrocortisone (1 µg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF(+) cells formed aligned cell-to-cell 'trains' when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32-80 years) or duration in eye bank corneal preservation medium prior to use (3-10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p < 0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.


Subject(s)
Endothelial Cells/cytology , Limbus Corneae/blood supply , Microvessels/cytology , Cadaver , Cell Differentiation , Cell Proliferation , Cell Separation/methods , Cells, Cultured , Flow Cytometry , Humans , Limbus Corneae/cytology
15.
Invest Ophthalmol Vis Sci ; 55(12): 7925-33, 2014 Nov 18.
Article in English | MEDLINE | ID: mdl-25406286

ABSTRACT

PURPOSE: We evaluated the angiographic features of the affected limbus in patients with pterygia and assessed limbal reconstruction outcomes after limbal-conjunctival autograft (LCA) transplantation in terms of vascular remodeling. METHODS: We studied prospectively 31 eyes of 31 patients who underwent pterygium excision and LCA transplantation; 28 eyes of 28 normal participants served as controls. Anterior segment indocyanine green angiography (ICGA) was performed for each participant preoperatively and at 1 week, and 1 and 3 months postoperatively. The perioperative angiographic features of the pterygium were compared to those in normal eyes. The structural changes of the marginal corneal vascular arcades (MCAs) and LCA were quantitatively assessed postoperatively in terms of vascular density and lacunarity. RESULTS: Deteriorated MCAs that extended beyond the pterygium head were observed in the pterygium group. The pterygium had a dual blood supply from the conjunctival and episcleral circulations. In terms of limbal reconstruction, the engorged reperfusion vessels arose from the adjacent episcleral vessels along the limbus at 1 week postoperatively. The reconstructed MCAs had begun to appear at 1 month postoperatively and became apparent 3 months postoperatively in 26 (83.9%) of 31 eyes of the pterygium group, resulting in a successful clinical outcome. Higher vascular density and lower lacunarity were measured in the limbus and the graft at 3 months than at 1 month (P < 0.001 for all), which indicated fine reorganization of the reconstructed vessels. CONCLUSIONS: The pterygium had a dual blood supply, and the remodeling of the affected limbus and LCA continued up to 3 months postoperatively.


Subject(s)
Conjunctiva/transplantation , Corneal Transplantation , Limbus Corneae/blood supply , Pterygium/surgery , Aged , Case-Control Studies , Corneal Transplantation/methods , Female , Fluorescein Angiography , Humans , Limbus Corneae/pathology , Limbus Corneae/surgery , Male , Middle Aged , Prospective Studies , Pterygium/pathology , Transplantation, Autologous
16.
PLoS Biol ; 12(7): e1001912, 2014 Jul.
Article in English | MEDLINE | ID: mdl-25051267

ABSTRACT

Schlemm's canal (SC) plays central roles in ocular physiology. These roles depend on the molecular phenotypes of SC endothelial cells (SECs). Both the specific phenotype of SECs and development of SC remain poorly defined. To allow a modern and extensive analysis of SC and its origins, we developed a new whole-mount procedure to visualize its development in the context of surrounding tissues. We then applied genetic lineage tracing, specific-fluorescent reporter genes, immunofluorescence, high-resolution confocal microscopy, and three-dimensional (3D) rendering to study SC. Using these techniques, we show that SECs have a unique phenotype that is a blend of both blood and lymphatic endothelial cell phenotypes. By analyzing whole mounts of postnatal mouse eyes progressively to adulthood, we show that SC develops from blood vessels through a newly discovered process that we name "canalogenesis." Functional inhibition of KDR (VEGFR2), a critical receptor in initiating angiogenesis, shows that this receptor is required during canalogenesis. Unlike angiogenesis and similar to stages of vasculogenesis, during canalogenesis tip cells divide and form branched chains prior to vessel formation. Differing from both angiogenesis and vasculogenesis, during canalogenesis SECs express Prox1, a master regulator of lymphangiogenesis and lymphatic phenotypes. Thus, SC development resembles a blend of vascular developmental programs. These advances define SC as a unique vessel with a combination of blood vascular and lymphatic phenotypes. They are important for dissecting its functions that are essential for ocular health and normal vision.


Subject(s)
Anterior Eye Segment/anatomy & histology , Animals , Anterior Eye Segment/growth & development , Cell Lineage , Endothelial Cells/physiology , Eye/blood supply , Homeodomain Proteins/biosynthesis , Limbus Corneae/blood supply , Lymphangiogenesis , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Morphogenesis , Phenotype , Tumor Suppressor Proteins/biosynthesis , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/physiology
17.
Exp Eye Res ; 125: 226-35, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24979218

ABSTRACT

The mouse eye has been used as a model for studies on the microanatomy of the outflow pathways but most of what is known comes from histological sections. These studies have focused mainly on the morphological features of the trabecular meshwork, Schlemm's canal and aqueous channels that link to the superficial episcleral vasculature. However, the anatomical architecture of the aqueous outflow vessels and their relationship to each other and to the general vascular circulation is not well understood. The aim of this study was to provide a detailed description of the microarchitecture of the aqueous outflow vessels and their relationship to the superficial limbal/episcleral vasculature throughout the entire limbus. The aqueous outflow vessels and blood and lymphatic vessels were imaged in PECAM-1 and LYVE-1 immunostained whole anterior segments of adult mice and three-dimensional (3-D) reconstructions of the optical sections were generated to reveal the aqueous, blood and lymphatic architecture. The arterial supply, venous drainage, organisation of perilimbal vasculature, collector channels/aqueous veins and the morphology of Schlemm's canal were revealed in their entirety and the relationships between these structures is described. Schlemm's canal was PECAM-1 positive but there was no affinity for the lymphatic marker LYVE-1. We show that Schlemm's canal is a continuous circular structure and more often seen as a single, broad, varicose vessel with short regions appearing as a plexus. Aqueous veins link Schlemm's canal to the superficial vasculature and there were no direct links seen between the canal and the lymphatic vessels.


Subject(s)
Anterior Eye Segment/anatomy & histology , Aqueous Humor/metabolism , Limbus Corneae/blood supply , Animals , Anterior Eye Segment/blood supply , Mice, Inbred C57BL , Microscopy, Confocal , Models, Animal
18.
Invest Ophthalmol Vis Sci ; 54(12): 7470-7, 2013 Nov 15.
Article in English | MEDLINE | ID: mdl-24159088

ABSTRACT

PURPOSE: To determine the metrics of the marginal corneal vascular arcades (MCA). METHODS: The MCA and filling pattern was investigated using indocyanine green dye angiography (ICGA) in the fellow eye of patients with treated unilateral keratitis. Images were acquired using a scanning laser ophthalmoscope. Five contiguous squares (100 pixels) were aligned beyond the inner row of vessels extending approximately 700 µm into the limbal region and spanning an arc length of approximately 4 mm of the peripheral cornea. Geometrical properties of the MCA were determined using programs written in a numerical computing environment. RESULTS: A total of 17 patients (24-88 years) were included. Filling of the inferior corneal quadrant occurred first, followed by superior, nasal, and temporal quadrants. Mean area of a vascular loop of the MCA was 11.87 × 10⁻³ mm² (SD: 10.44 × 10⁻³ mm²) skewed (2.20) toward smaller sizes. Mean circumference of a vascular loop was 422.5 µm (SD: 218.7 µm) with major and minor axes of 158.9 µm and 90.8 µm. There were five (SD: 1.8) branches per loop with a segment length of 89.5 um (SD 163.8 µm). Vessels were tortuous (mean 0.19, SD: 0.16) with a fractal number of 1.51 (0.12). There were significant differences between subjects in vessel loop area (P = 0.003) and number of branches (P = 0.002). Speed of flow was circumferential along the innermost row and measured at 0.22 mm/s in one subject. CONCLUSIONS: The MCA comprise a network of branched interlinked elliptical loops supporting circumferential blood flow in the corneal periphery. There was no definable change in vascular pattern extending into the limbal region.


Subject(s)
Ciliary Arteries/pathology , Corneal Neovascularization/physiopathology , Corneal Ulcer/physiopathology , Keratitis, Herpetic/physiopathology , Limbus Corneae/blood supply , Adult , Aged , Aged, 80 and over , Coloring Agents , Corneal Ulcer/therapy , Female , Fluorescein Angiography , Humans , Indocyanine Green , Keratitis, Herpetic/therapy , Male , Middle Aged , Observer Variation , Ophthalmoscopy , Regional Blood Flow , Sclera/blood supply , Young Adult
19.
Prog Retin Eye Res ; 34: 89-124, 2013 May.
Article in English | MEDLINE | ID: mdl-23348581

ABSTRACT

The cornea is one of the few tissues which actively maintain an avascular state, i.e. the absence of blood and lymphatic vessels (corneal [lymph]angiogenic privilege). Nonetheless do several diseases interfere with this privilege and cause pathologic corneal hem- and lymphangiogenesis. The ingrowths of pathologic blood and lymphatic vessels into the cornea not only reduce transparency and thereby visual acuity up to blindness, but also significantly increases the rate of graft rejections after subsequent corneal transplantation. Therefore great interest exists in new strategies to target pathologic corneal (lymph)angiogenesis to promote graft survival. This review gives an overview on the vascular anatomy of the normal ocular surface, on the molecular mechanisms contributing to the corneal (lymph)angiogenic privilege and on the cellular and molecular mechanisms occurring during pathological neovascularization of the cornea. In addition we summarize the current preclinical and clinical evidence for three novel treatment strategies against ocular surface diseases based on targeting pathologic (lymph)angiogenesis: (a) modulation of the immune responses after (corneal) transplantation by targeting pathologic (lymph)angiogenesis prior to and after transplantation, (b) novel concepts against metastasis and recurrence of ocular surface tumors such as malignant melanoma of the conjunctiva by anti(lymph)angiogenic therapy and (c) new ideas on how to target ocular surface inflammatory diseases such as dry eye by targeting conjunctival and corneal lymphatic vessels. Based on compelling preclinical evidence and early data from clinical trials the novel therapeutic concepts of promoting graft survival, inhibiting tumor metastasis and dampening ocular surface inflammation and dry eye disease by targeting (lymph)angiogenesis are on their way to translation into the clinic.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Cornea/blood supply , Corneal Neovascularization/drug therapy , Conjunctiva/blood supply , Cornea/physiology , Corneal Neovascularization/physiopathology , Humans , Limbus Corneae/blood supply , Lymphangiogenesis/physiology , Vascular Endothelial Growth Factors/antagonists & inhibitors , Vascular Endothelial Growth Factors/metabolism
20.
Optom Vis Sci ; 89(12): 1682-90, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23190713

ABSTRACT

PURPOSE: To estimate the prevalence of self-reported "sensitive eyes" (SEs) in soft contact lens (CL) wearers, evaluate the clinical characteristics of patients with SEs, and examine the effect of refitting them with silicone hydrogel lenses. METHODS: After self-assessment, 2154 CL wearers were separated into SE and non-SE patients. Demographics, biometric data, wearing time, symptoms, and signs were compared between the two populations. Sixty-three SE patients were randomized into senofilcon A (senA) lenses and 65 into a non-senA arm (lotrafilcon B, omafilcon A, and balafilcon A lenses). The performance of senA lenses was compared against habitual and non-senA lenses 2 weeks later. RESULTS: A total of 12.2% of CL wearers reported SEs with their habitual CLs. No significant differences were noticed between SE and non-SE patients in sex, age, or refraction. The prevalence of dryness (43 vs 19%, p < 0.0001), irritation (25 vs 11%, p < 0.0001), redness (20 vs 6%, p < 0.0001), and stinging (6 vs 1%, p < 0.0001) was higher in SE patients. Average wearing time (13.0 vs 14.1 hours, p < 0.0001) was lower in this group. Limbal/bulbar hyperemia and corneal/conjunctival staining were not significantly different between the two populations. Senofilcon A increased the number of patients reporting no dryness (habitual vs senA, 20 vs 44%, p < 0.0003), irritation (22 vs 37%, p = 0.015), redness (52 vs 76%, p =0.009) and stinging (58 vs 77%, p = 0.012) but did not significantly affect clinical signs. Senofilcon A was significantly more efficient than non-senA lenses in improving dryness (scale of 0 to 3: senA vs non-senA, 0.64 vs 1.02, p = 0.0056), irritation (0.72 vs 1.16, p = 0.0015), and stinging (0.18 vs 0.53, p = 0.0049). CONCLUSIONS: A substantial proportion of CL wearers report SEs with their habitual lenses. These patients are characterized by a high prevalence of additional symptoms, which are not reflected in clinical signs. Senofilcon A, or lenses with similar properties, may help reduce these symptoms in SE patients.


Subject(s)
Conjunctival Diseases/epidemiology , Contact Lenses, Hydrophilic/adverse effects , Corneal Diseases/epidemiology , Hyperemia/epidemiology , Limbus Corneae/blood supply , Adolescent , Adult , Conjunctiva/blood supply , Conjunctival Diseases/diagnosis , Conjunctival Diseases/etiology , Corneal Diseases/diagnosis , Corneal Diseases/etiology , Female , Follow-Up Studies , Humans , Hyperemia/diagnosis , Hyperemia/etiology , Male , Prevalence , Prosthesis Fitting , Refractive Errors/therapy , Surveys and Questionnaires , Young Adult
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