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Molecular Vision 2013To study the expression of S100 A and B family proteins in normal human limbus and to analyze modification of the expression in inflammatory conditions.
PURPOSE
To study the expression of S100 A and B family proteins in normal human limbus and to analyze modification of the expression in inflammatory conditions.
METHODS
The total expression of members of the S100 family and the expression of A4, A8, A9, and B individually were evaluated in nine normal human corneal limbi, collected from cadaver healthy donors, in particular in the limbal epithelial crypts (LECs), and in five inflamed limbi obtained from enucleated eyes. S100 protein distribution was determined with immunohistochemistry staining analysis.
RESULTS
Cytoplasmic expression of total S100 proteins was observed in 100% of LECs; in contrast, the inflamed tissues were completely negative, and faint positivity was observed in only one case. Moreover, cytoplasmic expression of S100 A4 and A9 was uniformly found in the entire LECs in all samples analyzed, while S100 A8 positivity was observed in only 44.4% of cases and only in the cells localized in the central area of the LEC. Positivity for S100 B was not observed in all samples analyzed.
CONCLUSIONS
As reported in the literature, normal limbal epithelial cells show strong expression of S100 proteins. A novel finding of this study was the expression for the limbal epithelial crypts. In particular, S100 A4 and A9, which are normally involved in regulating a wide range of biologic effects, including cell motility, survival, and differentiation, are the most expressed members in healthy limbal crypts. In inflamed tissues, expression of S100 proteins was dramatically decreased. S100 proteins, and in particular S100 A4 and S100 A9, can be useful as markers of early changes in stem cell niches due to inflammation.
Topics: Aged; Aged, 80 and over; Calgranulin A; Calgranulin B; Case-Control Studies; Cytoplasm; Genetic Markers; Humans; Inflammation Mediators; Keratitis; Limbus Corneae; Middle Aged; Nerve Growth Factors; S100 Calcium Binding Protein beta Subunit; S100 Calcium-Binding Protein A4; S100 Proteins; Stem Cell Niche
PubMed: 23378728
DOI: No ID Found -
American Journal of Ophthalmology Oct 2015To investigate the epithelial thickness in the cornea and limbus in limbal stem cell deficiency (LSCD) by using in vivo laser scanning confocal microscopy. (Comparative Study)
Comparative Study
PURPOSE
To investigate the epithelial thickness in the cornea and limbus in limbal stem cell deficiency (LSCD) by using in vivo laser scanning confocal microscopy.
DESIGN
Cross-sectional comparative study.
METHODS
Confocal images of 48 eyes of 35 patients with LSCD collected by the Heidelberg Retina Tomograph III Rostock Corneal Module Confocal Microscope from 2010 to 2014 were analyzed. Volume Z-scans of the central cornea and the superior, nasal, inferior, and temporal limbus were included in the analysis. Eleven normal eyes served as control. Epithelial thickness in all locations was measured by 2 independent observers.
RESULTS
The mean epithelial layer thickness was 48.6 ± 2.3 μm in the central cornea and 63.7 ± 11.3 μm in the limbus in the control. Compared with the epithelial thickness in normal control, the epithelial thickness in LSCD patients was reduced by an average of 20.2% in the central cornea and 38.5% in the limbus (all P < .05). The mean corneal epithelial thickness in patients with LSCD reduced 7.6%, 20.8%, and 61.3% in the early, intermediate, and late stage, respectively, compared to the control. In the limbus, the overall epithelial thickness decreased 30.0%, 39.7%, and 62.8% in the early, intermediate, and late stage of LSCD, respectively (all P < .05). Epithelial thinning correlated with the severity of LSCD in both cornea and limbus. In eyes with sectoral LSCD, a similar degree of epithelial thinning was also detected in the clinically unaffected limbal regions.
CONCLUSIONS
Both corneal and limbal epithelia become progressively thinner in LSCD. Epithelial thickness could be used as a diagnostic measure of LSCD.
Topics: Adult; Aged; Aged, 80 and over; Corneal Diseases; Cross-Sectional Studies; Epithelium, Corneal; Female; Humans; Limbus Corneae; Male; Microscopy, Confocal; Middle Aged; Organ Size; ROC Curve; Stem Cells; Young Adult
PubMed: 26163009
DOI: 10.1016/j.ajo.2015.06.029 -
The Ocular Surface Jan 2017Chemical injuries frequently result in vision loss, disfigurement, and challenging ocular surface complications. Acute interventions are directed at decreasing the... (Review)
Review
Chemical injuries frequently result in vision loss, disfigurement, and challenging ocular surface complications. Acute interventions are directed at decreasing the extent of the injury, suppressing inflammation, and promoting ocular surface re-epithelialization. Chronically, management involves controlling inflammation along with rehabilitation and reconstruction of the ocular surface. Future therapies aimed at inhibiting neovascularization and promoting ocular surface regeneration should provide more effective treatment options for the management of ocular chemical injuries.
Topics: Burns, Chemical; Eye Injuries; Humans; Limbus Corneae; Stem Cell Transplantation; Treatment Outcome
PubMed: 27650263
DOI: 10.1016/j.jtos.2016.09.002 -
Romanian Journal of Ophthalmology 2017The article presents the modern treatment with stem cells in the reconstruction of ocular surface. The turnover of the stem cells, the location in the limbus areas, the... (Review)
Review
The article presents the modern treatment with stem cells in the reconstruction of ocular surface. The turnover of the stem cells, the location in the limbus areas, the importance of limbal stem cells presence, the clinical appearance of stem cell deficiency, investigations method for this illness and the management of stem cell deficiency (artificial lacrimal tear drops, mini scleral contact lenses and the surgical treatment with allografts and autografts of stem cells) were taken into account.
Topics: Corneal Diseases; Epithelium, Corneal; Humans; Limbus Corneae; Regeneration; Stem Cell Transplantation; Stem Cells
PubMed: 29516041
DOI: No ID Found -
Indian Journal of Ophthalmology Mar 2004The past two decades have witnessed remarkable progress in limbal stem cell transplantation. In addition to harvesting stem cells from a cadaver or a live related donor,... (Review)
Review
The past two decades have witnessed remarkable progress in limbal stem cell transplantation. In addition to harvesting stem cells from a cadaver or a live related donor, it is now possible to cultivate limbal stem cells in vitro and then transplant them onto the recipient bed. A clear understanding of the basic disease pathology and a correct assessment of the extent of stem cell deficiency are essential. A holistic approach towards management of limbal stem cell deficiency is needed. This also includes management of the underlying systemic disease, ocular adnexal pathology and dry eye. Conjunctival limbal autografts from the healthy contralateral eye are performed for unilateral cases. In bilateral cases, tissue may be harvested from a cadaver or a living related donor; prolonged immunosuppression is needed to avoid allograft rejection in such cases. This review describes the surgical techniques, postoperative treatment regimes (including immunosuppression for allografts), the complications and their management. The short and long-term outcomes of the various modalities reported in the literature are also described.
Topics: Clinical Trials as Topic; Corneal Diseases; Epithelium, Corneal; Humans; Immunosuppressive Agents; Limbus Corneae; Postoperative Care; Stem Cell Transplantation; Tissue Preservation; Visual Acuity
PubMed: 15132374
DOI: No ID Found -
The British Journal of Ophthalmology Sep 2017To evaluate the efficacy of Fourier domain-optical coherence tomography (FD-OCT) in imaging and quantifying the limbal palisades of Vogt and to correlate these images...
AIMS
To evaluate the efficacy of Fourier domain-optical coherence tomography (FD-OCT) in imaging and quantifying the limbal palisades of Vogt and to correlate these images with histological findings.
METHODS
The superior and inferior limbal region of both eyes of 50 healthy volunteers were imaged by FD-OCT. Images were processed and analysed using Matlab software. In vitro immunofluorescent staining of a cadaveric donor limbus was analysed to correlate the presence of stem cells in the visualised structures.
RESULTS
FD-OCT could successfully visualise limbal crypts and the palisades of Vogt in the limbus region. Fluorescent labelling confirmed the presence of stem cells in these structures. The mean palisade ridge width (Δ) and the mean interpalisade epithelial rete peg width (Δ) were both of the order of 72 μm, leading to a palisade density (PD) of about . A significant difference in Δ, Δ and PD was seen between the inferior and superior sides of the right eye and the superior sides of the left and right eye(p<0.05.). A significant influence of iris colour on parameters Δ, Δ and PD was found, and of age on PD and Δ (p<0.05).
CONCLUSIONS
In vivo OCT imaging is a safe and effective modality to image the limbus and can be used to visualise the palisades of Vogt. Image processing using Matlab software enabled quantification and density calculation of imaged limbal palisades of Vogt. This technique may enhance targeted limbal biopsies for transplantation.
Topics: Adult; Epithelium, Corneal; Female; Fourier Analysis; Healthy Volunteers; Humans; Limbus Corneae; Male; Middle Aged; Stem Cell Niche; Tomography, Optical Coherence; Young Adult
PubMed: 28228408
DOI: 10.1136/bjophthalmol-2016-309549 -
Scientific Reports Aug 2019The role of the corneal epithelium and limbus in corneal avascularity and pathological neovascularization (NV) is not well understood. To investigate the contributions...
Application of corneal injury models in dual fluorescent reporter transgenic mice to understand the roles of the cornea and limbus in angiogenic and lymphangiogenic privilege.
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.
Topics: Animals; Corneal Injuries; Debridement; Disease Models, Animal; Epithelium, Corneal; Fluorescent Dyes; Limbus Corneae; Lymphangiogenesis; Mice, Transgenic; Neovascularization, Physiologic
PubMed: 31444394
DOI: 10.1038/s41598-019-48811-z -
Investigative Ophthalmology & Visual... Jan 2014To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions. (Comparative Study)
Comparative Study
PURPOSE
To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions.
METHODS
Limbal epithelial cells were isolated from 139 donors using 15 variations of three dissociation solutions. All culture conditions were compared to the baseline condition of murine 3T3-J3 feeders with xenobiotic (Xeno) keratinocyte growth medium at 20% O2. Five Xeno and Xeno-free media with increasing concentrations of calcium and epidermal growth factor (EGF) were evaluated at 5%, 14%, and 20% O2. Human MRC-5, dermal (fetal, neonatal, or adult), and limbal stromal fibroblasts were compared. Statistical analysis was performed on the number of maximum serial weekly passages, percentage of aborted colonies, colony-forming efficiency (CFE), p63α(bright) cells, and RT-PCR ratio of p63α/K12. Immunocytochemistry and RT-PCR for p63α, ABCG2, Bmi1, C/EBPδ , K12, and MUC1 were performed to evaluate phenotype.
RESULTS
Dispase/TrypLE was the isolation method that consistently showed the best yield, viability, and CFE. On 3T3-J2 feeders, Xeno-free medium with calcium 0.1 mM and EGF 10 ng/mL at 20% O2 supported more passages with equivalent percentage of aborted colonies, p63α(bright) cells, and p63α/K12 RT-PCR ratio compared to baseline Xeno-media. With this Xeno-free medium, MRC-5 feeders showed the best performance, followed by fetal, neonatal, adult HDF, and limbal fibroblasts. MRC-5 feeders supported serial passages with sustained high expression of progenitor cell markers at levels as robust as the baseline condition without significant difference between 20% and 5% O2.
CONCLUSIONS
The LSC function can be maintained in vitro under appropriate Xeno-free conditions.
Topics: Adult; Aged; Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Female; Follow-Up Studies; Humans; Immunohistochemistry; Limbus Corneae; Male; Mice; Middle Aged; Oxygen; Stem Cells; Xenobiotics
PubMed: 24030457
DOI: 10.1167/iovs.13-12517 -
Experimental Eye Research Apr 2016We studied the reproducibility and stability of limbal stem cell deficiency (LSCD) in mice following controlled injuries to the corneal and limbal epithelia. In one... (Review)
Review
We studied the reproducibility and stability of limbal stem cell deficiency (LSCD) in mice following controlled injuries to the corneal and limbal epithelia. In one method, corneal and limbal epithelia were entirely removed with a 0.5 mm metal burr. In the other, limbus to limbus epithelial removal with the burr was followed by thermal injury to the limbus. These two methods were compared with a previously published one. Unwounded corneas were used as control. The corneas were examined monthly for three months by slit lamp with fluorescein staining. Immunofluorescence staining for cytokeratin 12 and 8 on corneal wholemount and cross sections were performed to determine the phenotype of the epithelium. Mechanical shaving of the epithelium, with or without thermal injury, resulted in a reproducible state of LSCD marked by superficial neovascularization, reduce of keratin 12 expression and presence of goblet cells on the cornea. The phenotype was stable in 100% of the eyes up to at least three months. Thermal injury produced a more severe phenotype with more significant stromal opacification. These corneal injury models may be useful for studying the mechanisms leading to limbal stem cell deficiency.
Topics: Animals; Corneal Injuries; Corneal Neovascularization; Disease Models, Animal; Eye Burns; Limbus Corneae; Mice; Stem Cells
PubMed: 26607808
DOI: 10.1016/j.exer.2015.11.012 -
International Ophthalmology Oct 2022Limbo-keratoplasty enables visual improvement and limbal stem cell transplantation at the same. During follow-up, most grafts show vascularization of the limbus....
PURPOSE
Limbo-keratoplasty enables visual improvement and limbal stem cell transplantation at the same. During follow-up, most grafts show vascularization of the limbus. However, it is unclear whether vascularization is harmful due to immunologic effects or helpful to nourish the limbal stem cells and is therefore necessary for a clear graft. The aim of our study is to analyze the influence of graft vascularization on graft survival following homologous limbo-keratoplasty.
METHODS
In this retrospective study, we assessed all consecutive limbo-keratoplasties performed in our hospital. All eyes with suitable photo-documentation were included and divided into two groups (limbal stem cell deficiency and corneal dystrophy). We categorized the grade of vascularization (0, 1, 2, 3, 3b) and analyzed clear graft survival, recurrence of the underlying disease and the endothelial cell density (ECD) with regard to the reason for the graft. Event rates were estimated with the Kaplan-Meier method.
RESULTS
A total of 79 eyes with limbal stem cell deficiency and 15 with corneal dystrophies were analyzed. A high degree of graft vascularization had a tendency for better graft survival in limbal stem cell deficiency, whereas in corneal dystrophies, grafts with no vascularization had preferable outcomes. Recurrence-free graft survival was only seen in grade 1 and 3 vascularization in corneal dystrophies.
CONCLUSION
Vascularization of the limbus seems to have an impact on the long-term outcome of limbo-keratoplasty. The effect seems to be favorable in limbal stem cell deficiency and on recurrence rates in corneal dystrophies. However, the latter might be overshadowed by an unfavorable immunologic effect in corneal dystrophies where the baseline immunologic risk profile is commonly more favorable than in limbal stem cell deficiency.
Topics: Corneal Diseases; Corneal Dystrophies, Hereditary; Corneal Transplantation; Follow-Up Studies; Graft Survival; Humans; Keratoplasty, Penetrating; Limbus Corneae; Retrospective Studies; Scleral Diseases; Stem Cell Transplantation
PubMed: 35381896
DOI: 10.1007/s10792-022-02291-9