Search results for: aniridia-associated keratopathy
4 The Morphological Changes of POV in Diabetic Patients and Its Correlation with Changes in Corneal Epithelium, Corneal Nerve, and the Fundus in Using Vivo Confocal Microscopy
Authors: Ji Jiazheng, Wang Jingrao, Jin Xin, Zhang Hong
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Diabetes mellitus is a metabolic disease characterized by high blood sugar. A long-standing hyperglycemic state can lead to various tissue damage. Diabetic retinopathy is the most common and widely studied ocular complication and has become the leading cause of blindness in my country. At the same time, diabetes has profound clinically relevant effects on the cornea, leading to keratopathy and vision-threatening. The cornea is an avascular tissue and is sensitive to hyperglycemia, Keratopathy caused by diabetes is usually chronic, they are called diabetic keratopathy or diabetic neurotrophic keratopathy, leading to several diabetic corneal complications including delayed epithelial wound healing, recurrent erosions, neuropathy, loss of sensitivity. Corneal stem cell dysfunction in diabetic patients as an important influencing factor of diabetic keratopathy. The consequences of this condition are often underestimated. The limbus is located between the cornea and the sclera tissue. The limbal stroma consists of a series of radial elevations with fibrovascular centers known as palisades of Vogt (POV). Previous studies have shown that palisades of Vogt (POV), as the main site of limbal stem cells, plays an important role in the homeostasis of the corneal epithelium. Therefore, POV plays a vital role in the healing of corneal epithelial surgery and postoperative evaluation. IVCM can observe the condition of the corneal epithelium at the cellular level. It has profound significance and guidance for the evaluation of limbal and limbal stem cells. We have previously observed structural changes in POV in HSK and HZO patients on IVCM. At present, there have been reports involving limbal stem cell dysfunction in diabetic patients, but the specific pathogenesis is still unclear. However, there are no studies on POV morphological changes in patients with DM. Therefore, we performed statistics and compared the correlation between POV morphological changes and corneal epithelial basal cell density, corneal nerves, and length of disease in DM patients and normal humans using IVCM studies. At the same time, fundoscopy was used to observe the correlation between the thickness of RNFL and the thickness of GCC and POV in diabetic patients. And to observe the correlation between SVD, DVD and POV for research.Keywords: confocal microscopy, fundus, limbal stem cells, diabetes
Procedia PDF Downloads 893 In vitro Modeling of Aniridia-Related Keratopathy by the Use of Crispr/Cas9 on Limbal Epithelial Cells and Rescue
Authors: Daniel Aberdam
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Haploinsufficiency of PAX6 in humans is the main cause of congenital aniridia, a rare eye disease characterized by reduced visual acuity. Patients have also progressive disorders including cataract, glaucoma and corneal abnormalities making their condition very challenging to manage. Aniridia-related keratopathy (ARK), caused by a combination of factors including limbal stem-cell deficiency, impaired healing response, abnormal differentiation, and infiltration of conjunctival cells onto the corneal surface, affects up to 95% of patients. It usually begins in the first decade of life resulting in recurrent corneal erosions, sub-epithelial fibrosis with corneal decompensation and opacification. Unfortunately, current treatment options for aniridia patients are currently limited. Although animal models partially recapitulate this disease, there is no in vitro cellular model of AKT needed for drug/therapeutic tools screening and validation. We used genome editing (CRISPR/Cas9 technology) to introduce a nonsense mutation found in patients into one allele of the PAX6 gene into limbal stem cells. Resulting mutated clones, expressing half of the amount of PAX6 protein and thus representative of haploinsufficiency were further characterized. Sequencing analysis showed that no off-target mutations were induced. The mutated cells displayed reduced cell proliferation and cell migration but enhanced cell adhesion. Known PAX6 targets expression was also reduced. Remarkably, addition of soluble recombinant PAX6 protein into the culture medium was sufficient to activate endogenous PAX6 gene and, as a consequence, rescue the phenotype. It strongly suggests that our in vitro model recapitulates well the epithelial defect and becomes a powerful tool to identify drugs that could rescue the corneal defect in patients. Furthermore, we demonstrate that the homeotic transcription factor Pax6 is able to be uptake naturally by recipient cells to function into the nucleus.Keywords: Pax6, crispr/cas9, limbal stem cells, aniridia, gene therapy
Procedia PDF Downloads 2102 Effect of Retinoic Acid Treatment on the Retinoic Acid Signaling Pathway in a siRNA-Based Aniridia Limbal Epithelial Cell Model, in Vitro
Authors: Shao-Lun Hsu, Tanja Stachon, Fabian N. Fries, Zhen Li, Shuailin Li, Shanhe Liu, Berthold Seitz, Swarnali Kundu, Maryam Amini, Shweta Suiwal, Nóra Szentmáry
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Purpose: Congenital aniridia is characterized by PAX6 haploinsufficiency, and aniridia-associated keratopathy (AAK). In AAK, limbal stem cell deficiency and impaired wound healing are wiPurpose: Congenital aniridia is characterized by PAX6 haploinsufficiency and aniridia-associated keratopathy (AAK). In AAK, limbal stem cell deficiency and impaired wound healing are widely observed in patients, which might be associated with an imbalanced retinoic acid (RA) signaling pathway. In the previous studies, we demonstrated the relationship between PAX6 and the altered expression levels of key markers in the RA signaling pathway to retinol treatment. The present study evaluates the gene and protein expression levels in an in vitro small interfering RNA (siRNA) PAX6 knockdown aniridia limbal epithelial cell model following retinoic acid treatment. This study targets the direct effects of active RA products and their association with key regulators of the RA signaling pathway in siRNA PAX6 knockdown LECs. Methods: Primary human limbal epithelial cells (LECs) were knocked down by siRNA treatment to mimic PAX6 deletion in congenital aniridia (n=8). This was followed by 0µM, 1µM, and 5µM retinoic acid treatment applied in both siRNA PAX6 control and knockdown groups. After 48h incubation, paired box 6 (PAX6), alcohol dehydrogenase 7 (ADH7), aldehyde dehydrogenase 1 family member A1 (ALDH1A1), cytochrome P450 family 26 subfamilies A member 1 (CYP26A1), retinol-binding protein 1 (RBP1), cellular retinoic acid binding protein 2 (CRABP2), fatty acid binding protein 5 (FABP5), retinoid X receptor alpha (RXRA), retinoid X receptor beta (RXRB), retinoic acid receptor alpha (RARA), retinoic acid receptor beta (RARB), peroxisome proliferator-activated receptor gamma (PPARG), vascular endothelial growth factor A (VEGFA) mRNA levels have been determined using qPCR and protein levels by ELISA or western blot. Results: PAX6, ADH7, ALDH1A1, FABP5 mRNA levels and PAX6, ADH7, FABP5, PPARG2 protein levels were significantly lower in the PAX6 knockdown group, than in controls (p<0.001, p=0.018, p=0.015, p<0.001; p<0.001, p=0.003, p<0.001, p=0.007). PPARG mRNA level was significantly higher in the PAX6 knockdown group than in controls (p=0.012). CYP26A1 mRNA expression was upregulated using 1 µM and 5 µM RA treatment in both the PAX6 control (p<0.001; p<0.001) and the PAX6 knockdown group (p=0.001; p=0.002). CRABP2 mRNA expression in the PAX6 knockdown group (p=0.02) and protein expression in both groups were downregulated using to 5 µM RA concentration (p=0.003; p=0.02). RARA mRNA expression in the PAX6 knockdown group (p=0.023), RARB mRNA expression in both groups (p=0.006, p=0.001), and RXRA protein expression in controls (p=0.007), were downregulated using 5 µM RA concentration. VEGFA mRNA expression in PAX6 controls was upregulated using 5 µM RA (p=0.041). FABP5 to CRAP2 ratio was higher in PAX6 controls than in the PAX6 knockdown group (p<0.001). Additionally, the FABP5 to CRAP2 ratio was only upregulated in PAX6 controls using 5 µM RA concentration but not in the PAX6 knockdown group (p<0.001). Conclusions: These results reveal a less-responsive FABP5 to CRABP2 ratio in PAX6 knockdown LECs following increased RA concentration, as well as altered expression of key regulators in the RA signaling pathway. Further investigations into the regulatory processes are required to elucidate the role of RA signaling in the development of AAK.Keywords: congenital aniridia, paired box 6 gene, aniridia-associated keratopathy, retinoic acid signaling pathway
Procedia PDF Downloads 01 Outcomes of Combined Penetrating keratoplasty and Vitreo-Retinal Surgery in Management of Endophthalmitis with Obscured Corneal Clarity
Authors: Abhishek Dave, Manisha Singh
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Purpose: The study aims to evaluate the outcomes of combined Penetrating keratoplasty (PKP) and Vitreo-Retinal (VR) surgery in patients having endophthalmitis with poor corneal clarity. Methods: PKP with VR Surgery was performed in 43 eyes. This is a retrospective analysis of their preoperative, intraoperative and microbiological characteristics and anatomical and functional outcomes. Results: Corneal opacification was due to corneal ulcer in 30 (69.7%), graft infection in 8 (18.6%), bullous keratopathy in 4 and corneal scar in 1 eye. Postoperative visual acuity improved in 20 (46.5%), not changed in 14 (32.5%) and deteriorated in 9 eyes (20.9%). Poor anatomic success was seen in 15 (34.88%) eyes (9-phthisis bulbi, 6-eviscerated). Graft remained clear in 24 eyes (1 year). Microbiology revealed bacteria in 26, fungus in 14 and no growth in 3 eyes. Six out of 11 patients having poor vision in the fellow eye, too, achieved functional success. Conclusion: PKP with VR surgery is a complex but globe-salvaging procedure for poor prognosis eyes, which otherwise would need evisceration.Keywords: penetrating keratoplasty, VR surgery, endophthalmitis, corneal ulcer
Procedia PDF Downloads 54