Molecular Vision最新文献

Purpose: Intravitreal injection of adeno-associated virus (AAV) vectors is a good approach for transducing retinal ganglion cells (RGCs) in mice. It allows for high transduction efficiency and is relatively specific to RGCs. To deliver vectors, most studies use a transscleral approach that can have potentially negative effects, causing damage to the lens or retina. We optimized the intravitreal injection method using a transpupillary approach to minimize ocular damage and efficiently transfect RGCs.

Methods: C57BL/6J mice were anesthetized, and their irises were dilated. The eyeball was held with forceps while a small, full-thickness incision was made halfway between the center and periphery of the cornea. Using a bent 35-gauge blunt needle, the tip was navigated through the incision across the anterior chamber to reach the distal aspect of the pupil. The needle was inserted through the pupil, swept around the lens, and entered the vitreous, delivering expression vectors containing cytomegalovirus (CMV) promoter-driving green fluorescent protein (AAV-CMV-GFP) into the vitreous chamber. Fourteen days after injection, live fluorescent fundus images were taken, followed by immunostaining for GFP.

Results: With the improved injection technique, the lens remained clear and undamaged. Fundus imaging and GFP staining showed that over 90% of the mouse retinas sustained no visible damage. Retinas injected via the transpupillary approach also exhibited GFP transduction throughout the ganglion cell layer.

Conclusions: Transpupillary intravitreal injection reduces the potential risk compared to the transscleral approach, offering a promising and efficient method for delivering reporter genes to RGCs and ensuring high levels of gene expression without damage to the lens or retina.

Purpose: To date, the assessment of lens epithelial cell viability has been proposed only in cell cultures or isolated capsule models. This study aimed to develop a method for quantifying the viability of epithelial cells on whole ex vivo crystalline lenses by triple labeling Hoechst 33342, ethidium homodimer, and calcein-acetoxymethyl (HEC).

Methods: Two models of induced cell death were used to study the performance and potential applications of the technique. First, ten fresh pairs of six-month-old porcine lenses were retrieved. On one lens of each pair, an easily identifiable localized lesion was induced by a calibrated cryo-application, while the other remained intact. Both lenses of each pair were incubated for 1 h at 20 °C in an HEC mixture. Ten other pairs of lenses were used in the second experiment. On one lens of each pair, a diffuse epithelial lesion was induced by incubation in staurosporine (STS) solution (0.5 µM in CorneaMax) for 24 h at 37 °C. The other lens of each pair was incubated in CorneaMax solution without STS for 24 h at 37 °C. The day after, both lenses of each pair were incubated for 1 h at 20 °C in an HEC mixture. Images were acquired with a macroscope (macro zoom) and analyzed with ImageJ. Calcein-AM and ethidium images were used to calculate the area covered by living epithelial cells. Hoescht images allowed us to count cell nuclei per unit area. Viable epithelial cell density (vECD) was defined as the number of viable cells per unit area. Different strategies were developed to reduce background noise.

Results: There was no interfering lens autofluorescence for the exposure times used. The vECD median was 2,840 cells/mm² [10th-90th percentiles = 2,479-3,494] for cryo-injured lenses versus 3,364 cells/mm² [2,919-3,739] for healthy lenses (p = 0.002). The vECD median was 3,804 cells/mm² [10th-90th percentiles = 2,922-4,862] for lenses treated with STS versus 3,896 cells/mm² [3,169-4,980] for healthy lenses (p = 0.002).

Conclusions: Thanks to simple sample preparation, triple HEC staining allows fluorescence imaging of a large series of a whole lens to respect the architecture of the epithelium. It will be particularly useful for cytotoxicity studies of new therapies targeting the lens.

Purpose: This study identified the genetic causes of Axenfeld-Rieger syndrome (ARS) in a Chinese family and evaluated their clinical phenotype and clinical treatment.

Methods: We recruited a Chinese family with ARS. The proband presented with bilateral ectopic pupils, periumbilical redundancy, craniofacial abnormalities, and dental abnormalities after birth and was diagnosed with ARS. The symptoms were the same for her younger brother. Blood samples were collected from four family members: the proband, her brother, and her parents. Whole-genome sequencing (WGS) was performed to identify probable genetic variants in the proband. To confirm the identified variants, samples from the other family members were subjected to quantitative polymerase chain reaction (qPCR) and Sanger sequencing.

Results: Based on the results of WGS, we suspected a deletion region and an inversion region around the PITX2 gene. Through qPCR and Sanger sequencing, we identified a complex rearrangement involving a 6.15 Mb deletion on Chromosome 4, including the PITX2 coding region (Hg38; chr4:110617776-116769011), a 45.71 Mb inversion (Hg38; chr4:116769011-162481408), and a 14-bp deletion (Hg38; chr4:162481409-162481422). Interestingly, the father's copy number was normal, but Sanger sequencing revealed the same breakpoints. This indicated that the father is a balanced rearrangement carrier, and the children are unbalanced rearrangement carriers. While similar deletions and many breakpoints in this region have been reported, this specific rearrangement is novel.

Conclusions: Using WGS, qPCR, and Sanger, we found a complex genomic rearrangement with the deletion of PITX2 in a Chinese family with ARS. The clinical characteristics of the affected individuals were reported. The current findings broaden our understanding of the phenotype and variant spectrum associated with ARS caused by PITX2 deletion.

Purpose: This study aimed to investigate the association between proton pump inhibitors and dry eye disease (DED) among hospitalized SARS-CoV-2 patients.

Methods: We conducted a retrospective cohort study using electronic medical records from patients hospitalized for SARS-CoV-2 between April 2020 and December 2023. Eligible participants were aged over 18 years and hospitalized for SARS-CoV-2 without preexisting DED. Exclusions included ICU admissions, malignancies, recent ocular interventions, or chronic medications known to induce dry eye disease. Logistic regression adjusted for age, gender, and vaccination status evaluated associations between gastrointestinal (GI) medications and the subsequent development of dry eye disease within 6 months following hospital discharge.

Results: The age and gender distributions within the cohort were representative of the general SARS-CoV-2 infected population. Among 1165 patients, 167 (14.3%) developed dry eye disease post-hospitalization. Laxative use (lactulose and polyethylene glycol) correlated positively with dry eye disease (OR 1.939, p = 0.016; OR 2.094, p = 0.015, respectively). Metoclopramide treatment showed the strongest association (OR 13.413, p < 0.001), with over 50% incidence in affected patients. Conversely, omeprazole showed an inverse correlation with dry eye disease (OR 0.332, p < 0.001). Polypharmacy increased the odds of DED (odds ration [OR] 1.629, p = 0.015), while age, gender, and vaccination status did not significantly influence the outcomes.

Conclusions: Our findings emphasize significant correlations between GI medications and dry eye disease in SARS-CoV-2 survivors. Proton pump inhibitors may mitigate the risk of dry eye disease, contrasting with adverse effects linked to laxatives and metoclopramide. Vasoactive intestinal peptide (VIP), which links gut and lacrimal gland functions, is a strong candidate for the basis of the underlying pathophysiological mechanisms.

Purpose: During ocular trauma, excessive proliferation and transdifferentiation of corneal stromal fibroblasts cause haze/fibrosis in the cornea. Transforming growth factor β (TGFβ) plays a key role in corneal fibrosis through the Smad signaling pathway. The aberrant activity of TGFβ signaling during ocular trauma (viz. mechanical, infectious, chemical, or surgically altered TGFβ/Smad signaling) leads to regulating the predominant expression of myogenic proteins and the extracellular matrix (ECM). We sought to investigate the functional role of Smad3 in corneal wound repair and stromal ECM assembly using Smad3^+/+ wild-type and Smad3^-/- deficient mice.

Methods: Corneal injury was introduced with the topical application of an alkali-soaked 2-mm filter disc on the central cornea in the Smad3^+/+ (C57BL/6J) and Smad3^-/- (129-Smad3^tm1Par/J) mouse strains. Slit-lamp and stereo microscopy were used for clinical assessment and corneal haze grading in live animals. Hematoxylin and eosin and Masson's trichrome staining were used to study comparative morphology and collagen level alterations between the groups. Real-time qRT-PCR, western blot, and immunohistochemistry were used to measure changes in profibrotic genes at the mRNA and protein levels.

Results: Slit-lamp clinical exams and stereo microscopy detected notably less opaque cornea in the eyes of Smad3^-/- compared with Smad3^+/+ mice at 3 weeks (p<0.01) in live animals. Corneal tissue sections of Smad3^-/- mice showed significantly fewer α-smooth muscle actin-positive cells compared with those of the Smad3^+/+ animals (p<0.05). The corneas of the Smad3^-/- mice showed significantly lower mRNA levels of pro-fibrotic genes, α-smooth muscle actin, fibronectin, and collagen I (p<0.05, p<0.01, and p<0.001). In addition, the matrix metalloproteinase and tissue inhibitors of metalloproteinase levels were significantly increased (p<0.001) in the corneal tissue during alkali injury in both Smad3^+/+ wild-type and Smad3^-/- deficient mice.

Conclusions: The significant changes in profibrotic genes and stromal ECM proteins revealed a direct role of Smad3 in stromal ECM proteins and TGFβ/Smad-driven wound healing. Smad3 appears to be an attractive molecular target for limiting abnormal stroma wound healing to treat corneal fibrosis in vivo.

Background: Age-related macular degeneration (AMD) is a complex condition involving multiple factors. The condition is associated with numerous inflammatory indicators, including cytokines. Single-nucleotide polymorphisms in cytokine genes can also modify gene expression, perhaps contributing to the development of the disease. The objective of the present study was to examine the correlation among IL-6 SNPs (rs1800795, rs1800796, and rs1800797) and the serum levels of IL-6 in AMD patients treated at the Regional Institute of Ophthalmology of Pt. B.D. Sharma PGIMS, Rohtak (Haryana), India.

Methods: This case-control study included 131 patients diagnosed with AMD using precise ophthalmic examinations, such as slit lamp examination, fundoscopy, and ocular coherence tomography. To provide a basis for comparison, we also enlisted 100 healthy individuals as controls. Serum IL-6 protein levels were measured in both patients and controls using an enzyme-linked immunosorbent assay kit (ELISA). Genotyping IL-6 SNPs was performed using the PCR and DNA Sanger sequencing technique.

Results: IL-6 serum levels were considerably elevated in individuals with AMD compared to the control group (p < 0.05). Statistically significant differences were seen in the genotype frequencies of rs1800795 (p = 0.027) and rs1800797 (p = 0.0011) among the AMD patients and the healthy controls. Furthermore, strong correlations were observed between rs1800795 and the likelihood of developing AMD based on the heterozygous (OR = 2.04; p = 0.025), dominant (OR = 1.80; p = 0.035), and over-dominant models (OR = 2.10; p = 0.0094). Additionally, there were notable associations between rs1800797 and vulnerability to AMD through heterozygous (OR = 3.21; p = 0.009), dominant (OR = 2.74; p = 0.004), and over-dominant (OR = 3.11; p = 0.002) models. The rs1800795, rs1800796, and rs1800797 haplotypes C-G-A and G-G-A were linked to an elevated risk of AMD (p = 0.005, p = 0.024. respectively).

Conclusions: Our findings indicated a significant elevation in IL-6 serum levels among the AMD patient group compared to the control group. The interleukin-6 gene polymorphisms rs1800795 and rs1800797 were linked to an elevated risk of AMD in our study population.

Purpose: The purpose of this cross-sectional study was to investigate the relationship between oxidation balance scores (OBSs) and myopia.

Methods: Participant information came from the National Health and Nutrition Examination Survey (NHANES) 2007-2008. The relationship between OBSs and myopia was analyzed using a restricted cubic spline, generalized linear regression, trend analysis, and ordinal logistic regression. The important components of the OBS in myopia were analyzed using XGBoost, random forest, and AdaBoost.

Results: The data of 5,187 participants from NHANES were collected, and a preliminary analysis was conducted. We found that an association between OBSs and myopia was only found in participants aged ≥ 20 years (n = 4,253). There was a linear relationship between OBSs and the occurrence of myopia in them. The logistic regression showed that OBSs were correlated with an increased incidence of myopia after adjusting for all confounders (OR: 1.01, 95% CI [1.00, 1.02]). The trend test showed that the higher the OBS, the higher the likelihood of developing myopia (p for trend < 0.05). There was a nonlinear relationship between OBSs and myopia severity according to a generalized additive model (β = 0.01, 95% CI [0.00, 0.01], p < .01). The ordered logistic regression analysis showed that for every unit increase in OBS, the likelihood of myopia severity increased by 11% after adjusting for all confounders. We also found that calcium was an important OBS component related to the incidence of myopia.

Conclusions: OBS is positively associated with the occurrence and severity of myopia in adults ≥ 20 years of age, and calcium is an important OBS component related to the incidence of myopia.

Purpose: This study aimed to determine the role of peroxisome proliferator-activated receptor α (PPARα) on corneal epithelial wound healing.

Methods: Ten-week-old PPARα knockout (PPARα^-/- ) mice and wild-type (WT) C57BL/6 mice and ex vivo cultured human corneal epithelial cells were used to investigate the function of PPARα on corneal epithelial wound healing. A two-millimeter diameter of the mice's central corneal epithelium was removed to induce corneal epithelial injury. The expression of PPARα during corneal epithelial wound healing was analyzed using immunofluorescent staining and quantitative RT-PCR. Histological and immunostaining techniques were used to evaluate corneal morphology, cell proliferation, and inflammatory response in WT and PPARα^-/- mice. PPARα agonist fenofibrate was used to determine its effect on corneal epithelial wound healing.

Results: PPARα expression was found to significantly increase during corneal epithelial repair. PPARα^-/- mice exhibited delayed corneal epithelial wound healing compared to WT mice. PPARα^-/- mice displayed altered proliferative responses and distinct patterns of inflammatory infiltrates. Administration of fenofibrate to WT mice resulted in accelerated corneal epithelial repair and increased PPARα expression and cell proliferation. In vitro studies using human corneal epithelial cells further supported the impact of fenofibrate on promoting corneal epithelial cell wound healing.

Conclusions: PPARα is a regulator of corneal epithelial wound healing, and its absence leads to delayed repair processes in the corneal epithelium.

Purpose: North Carolina macular dystrophy (NCMD) is a rare autosomal dominantly inherited congenital maculopathy caused by either non-coding point mutations or tandem duplications in the DNase I hypersensitivity site DHS6S1, at chromosome 6q16 (MCDR1), or at chromosome 5 (MCDR3). To date, at least 30 NCMD pedigrees from different ethnicities have been genetically identified worldwide. Herein, we report the clinical and genetic features of a newly found NCMD family in Mexico with a novel tandem duplication involving both the DNASE1 site and the PRDM13 gene.

Methods: Seven affected subjects from a Mexican family underwent a complete ophthalmic assessment that included dilated indirect ophthalmoscopy, fundus photography, optical coherence tomography (OCT), fundus autofluorescence (FAF), kinetic and chromatic perimetry, and electroretinography (ERG). Next-generation sequencing (NGS), followed by array-based comparative genomic hybridization (array-CGH) and quantitative polymerase chain reaction (qPCR) analyzes, were employed to demonstrate the causative molecular defect.

Results: All seven affected patients had a severe appearing phenotype characterized by symmetric excavated atrophic coloboma-like chorioretinal macular lesions. In addition, using OCT, lacunae in the inner retinal layers and inner retinal loss were observed in all patients. NGS identified a heterozygous tandem duplication of the entire coding sequence of the PRDM13 gene in all seven affected individuals, whereas subsequent array CGH, NGS, and Sanger sequencing allowed for the identification of the precise boundaries of a ~148 kb MCDR1 duplication containing the whole PRMD13 gene and the DNASE1 site.

Conclusions: The phenotypic features in this NCMD pedigree continue to support the concept that this disorder is a congenital macular malformation rather than a progressive dystrophic entity. Unlike most NCMD families, there was no variable expressivity found in this study, possibly due to the relatively small size of the family. The other hypothesis is that the duplication involves genomic segments that are more consistently or tightly bound to other regulatory regions of PRDM13. The identification of a novel causative tandem duplication involving the DNASE1 site and the PRDM13 gene in this family allows for the expansion of the mutational spectrum of the disease.

Purpose: Diabetic patients experience chronic hyperglycemia that increases oxidative stress by enhancing free radical formation and nitric oxide (NO) production. Genetic mutations in the endothelial nitric oxide synthase (eNOS) enzyme gene affect the levels of NO formation. These mutations, together with chronic hyperglycemia, may increase the risk of diabetic retinopathy (DR) development and/or DR progression as a complication of diabetes. This study aimed to determine whether the eNOS polymorphisms intron 4ab, exon 7 Glu298Asp variant (G894T), and T-786C are associated with DR severity.

Methods: This case-control study involved 250 subjects (172 with type 2 diabetes mellitus (DM) with or without DR and 78 healthy controls). DR was detected by slit lamp biomicroscopy and its severity was determined with the evidence-based International Clinical Diabetic Retinopathy Disease Severity Scale. The genotyping of eNOS polymorphisms was analyzed by polymerase chain reaction (PCR) only or with restriction fragment length polymorphism. The haplotype analysis was performed using the SNPStats tool.

Results: The genotype distribution for the three polymorphisms was significantly different in patients with diabetes compared to controls (intron 4 ab: a/a, 1.7%; a/b, 20.4%; b/b, 77.9%. G894T: GG, 56.4%; GT, 34.3%; TT, 9.3%. T-786C: TT, 58.2%; TC, 33.5%; CC, 8.3%). Differences in genotype or allele frequency was non-significant between subjects with diabetes and DR compared to those without DR, except the C allele of the T-786C polymorphism was significantly less common in DR patients. DR severity was not affected by any polymorphisms. Haplotypes bTC and aTT were significantly less common or more prevalent in DR than DM patients, respectively.

Conclusions: We demonstrated that type 2 DM patients exhibited a higher prevalence of the three polymorphisms when compared to the control group. The C allele of T-786C polymorphism may have a protective effect against DR. Also, none of the mutations was correlated with a higher DR risk nor with the severity of DR. Haplotype aTT increased the risk for DR, while the bTC haplotype reduced it.