The Effect of Irradiated Riboflavin in Human Tenon's Fibroblast - A Study on Cellular Viability.

PURPOSE/ AIM: The main purpose of this work is to study the cellular viability effect of irradiated riboflavin in cultured human tenon fibroblasts. MATERIALS AND METHODS: The tenon tissue was harvested from a patient undergoing strabismus surgery. The human tenon fibroblast cell culture and isolation were performed according to the standard laboratory cell culturing protocol. The cells were divided into three groups: control, treatment with irradiated and non-irradiated riboflavin. There were five different concentrations (0.00156%, 0.003125%, 0.00625%, 0.0125%, 0.025%) in each group of riboflavin. The fibroblasts were treated with riboflavin and the cellular viability was assessed at 24-hour and 48-hour post treatment with MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide colorimetric assay. The absorbance values were analysed using Magellan microplate reader data analysis. A triplicate of readings was taken. The data were presented as mean ± standard deviation of the triplicates. Statistical analysis was performed with Statistical Package for Social Sciences (SPSS) analysis version 23. RESULTS: Irradiated riboflavin caused a concentration-dependent cell death in human tenon fibroblast cell culture (p < .05). The antiproliferative difference between irradiated and non-irradiated riboflavin was significant up to 48 hours (p < .05). Post hoc multiple comparisons showed higher concentrations of irradiated riboflavin (0.0125% and 0.025%) caused more reduction in cellular viability in human tenon fibroblast cells (p < .05). The duration of treatment is not a causative factor in this study. CONCLUSIONS: This pilot experiment demonstrated that irradiated riboflavin induced cell death in human tenon fibroblast culture in a concentration-dependent manner, but is not time-dependent. Further exploratory investigations should be performed to determine the mechanism of cell death. We postulate that apoptosis occurred in these irradiated riboflavin-treated cells.

Identification of potential serum metabolic biomarkers for patient with keratoconus using untargeted metabolomics approach.

This study aimed to investigate the metabolite differences between patients with keratoconus and control subjects and identify potential serum biomarkers for keratoconus using a non-targeted metabolomics approach. Venous blood samples were obtained from patients with keratoconus (n = 20) as well as from age-, gender- and race-matched control subjects (n = 20). Metabolites extracted from serum were separated and analyzed by liquid chromatography/quadrupole time-of-flight mass spectrometer. Processing of raw data and analysis of the data files was performed using Agilent Mass Hunter Qualitative software. The identified metabolites were subjected to a principal component and hierarchical cluster analysis. Appropriate statistical tests were used to analyze the metabolomic profiling data. Together, the analysis revealed that the dehydroepiandrosterone sulfate from the steroidal hormone synthesis pathway was significantly upregulated in patients with keratoconus (p < 0.05). Also, a combination of eicosanoids from the arachidonic acid pathway, mainly prostaglandin F2α, prostaglandin A2, 16,16-dimethyl prostaglandin E2, and 5-hydroxyeicosatetraenoic acid were collectively up-regulated as a group in keratoconus patients (p < 0.05). On the other hand, glycerophospholipid PS(17:2(9Z,12Z)/20:4(5Z,8Z,11Z,14Z)) was found to be significantly upregulated in the metabolomics profiles of control subjects (p < 0.05). The differently regulated metabolites provide insights into the pathophysiology of keratoconus and could potentially be used as biomarkers for keratoconus to aid in screening for individuals at risk hence, enabling early diagnosis and timely monitoring of disease.

Intravitreal bevacizumab versus ranibizumab: Effects on the vessels of the fellow non-treated eye.

PURPOSE: To evaluate the effects of anti-vascular endothelial growth factors (anti-VEGF) on the vessels of the non-treated eyes following intravitreal injections. METHODS: In this prospective, non-randomized trial, a total of 38 patients were recruited. 21 patients received ranibizumab, and 17 patients received bevacizumab. Fundus photography was carried out at baseline immediately before injection and at 3 days, 7 days, and one month after the injections. Using image analysis software, measurements were summarized as the central retinal artery equivalent (CRAE) and central retinal vein equivalent. RESULTS: In non-treated eyes, CRAE decreased significantly from 153.23 ± 15.20 µm before injection to 148.77 ± 17.21 µm 3 days after intravitreal bevacizumab (P = 0.004). There was no significant difference in CRAE, between the pre-injection baseline, one week, and one month after intravitreal bevacizumab injection in non-treated eyes (P > 0.05). No significant difference was noted in CRAE in the non-treated eyes of the ranibizumab group at any post-injection visit (P = 0.1). CONCLUSION: A significant transient narrowing effect of bevacizumab on retinal arterioles in the fellow non-treated eyes on the third day after intravitreal injection may show that plasma concentrations of these drugs are sufficient to spread the effect to the other eye.

Dosage-dependent reduction of macular pigment optical density in female breast cancer patients receiving tamoxifen adjuvant therapy.

It is now increasingly common for breast cancer patients to receive adjuvant tamoxifen therapy for a period of up to 10 years. As survival rate increases, managing tamoxifen ocular toxicities is important for patients' quality of life. Macular pigments in photoreceptor cells protect against free radical damage, which can cause macular degeneration. By reducing macular pigment concentration, tamoxifen may increase the risk of macular degeneration. Here, we compared macular pigment optical density (MPOD) and central macular thickness between breast cancer patients on tamoxifen adjuvant therapy (n = 70), and a control group (n = 72). Multiple regression analysis indicated that MPOD decreases with increasing tamoxifen dosage, up to a threshold of about 20 g, after which MPOD plateaus out. Mean MPOD in the treatment group (mean = 0.40) was significantly lower (p-value = 0.02) compared to the control group (mean = 0.47) for the left eye, and for the right eye (treatment mean = 0.39; control mean = 0.48; p-value = 0.009). No significant difference in mean central macular thickness was found between the treatment and the control group (p-values > 0.4). In the control group, MPOD and central macular thickness showed significant correlation (r∼0.30; p-values < 0.01) for both eyes. However, in the treatment group, loss of significant correlation was observed in the left eye (r = 0.21; p-value = 0.08). The present results show that MPOD decreases non-linearly as a function of tamoxifen dosage, and highlight the potential of tamoxifen to reduce macular pigment concentration through an unknown mechanism that does not depend on macular thinning solely.

Role of Angiography in Systemic Lupus Erythematosus-Induced Choroiditis.

PURPOSE: To report a case of systemic lupus erythematosus-induced choroidal vasculitis. METHODS: A 34-year-old woman with a long-standing history of systemic lupus erythematosus had a sudden painless loss of vision in the right eye over 12 hours. Ocular examination revealed a visual acuity of counting fingers of 1 foot on the right eye and 20/20 on the left. There was a relative afferent pupillary defect on the right side with a pink, distinct optic disk margin. RESULTS: Optical coherence tomography of the macula and fundus fluorescein angiogram for the eyes were normal. The MRI brain and orbit with the cerebral MRA did not show signs of optic neuritis or occipital vasculitic changes. However, the indocyanine green angiography revealed patches of ill-defined areas of choroidal hypofluorescence in the early- to mid-phase in the macula region. CONCLUSION: ICGA becomes the crucial tool in unmasking the presence of choroidal vasculitis.

Diabetes and pachymetry changes in pregnancy.

BACKGROUND: Purpose of this study is to evaluate changes in the central corneal thickness (CCT) in patients during the third trimester and postpartum phases of normal pregnancy, pregestational diabetes mellitus (DM), and gestational diabetes mellitus (GDM). METHODS: This was a prospective study. Patients that fulfilled the inclusion criteria were recruited from the obstetric clinic. They were grouped into normal pregnancy, pregestational DM, and GDM. Ophthalmic assessment and haematological investigations were done during the third trimester (after 28 weeks of gestation) and in the postpartum phase (6 weeks postpartum). RESULTS: A total of 192 pregnant patients were recruited for this study. Out of the 192 patients, only 143 of them came back for their follow-up 6 weeks postpartum. A total of 70 (36.5%) normal pregnancy patients, 51 (26.6%) DM patients, and 71 (36.9%) GDM patients were included in this prospective study. Our study showed that the CCT decreased postpartum in all three groups. Patients in all three groups did not have significantly different CCT during the third trimester of pregnancy and postpartum phase. However, patients who had thicker CCT irrespective of the grouping during the third trimester also had a thicker CCT post-delivery (p value <0.001). However, these changes did not appear to affect refractive error and visual acuity. CONCLUSIONS: Diabetes mellitus during pregnancy did not appear to influence the CCT.

Association of copy number variations in complement factor H-Related genes among age-related macular degenerative subjects.

Age-related macular degeneration (AMD) is the most widely recognised cause of irreversible vision loss and previous studies have suggested that the advancement of wet AMD is influenced by both modifiable and non-modifiable elements. Single nucleotide polymorphism (SNPs) and copy number of variations (CNVs) have been associated with AMD in various populations, however the results are conflicting. Our aim is to determine the CNVs of Complement Factor H-Related genes among Malaysian subjects with wet AMD. 130 patients with wet AMD and 120 healthy controls were included in this research. DNA was extracted from all subjects and CNVs of CFH, CFHR1 and CFHR3 genes; determined using quantitative real-time PCR and were compared between the two groups. A consistent association was observed between CFH gene and wet AMD susceptibility (P < 0.05). The age-adjusted data suggests a possible increased risk of AMD disease (P < 0.05). No correlation was detected between CNVs and wet AMD for the remaining genes after we compared the frequencies of mean for that gene. An association was observed between CFH CNVs and wet AMD in the Malaysian population, however, strong evidence of a link with wet AMD was not found. Further investigative studies are needed using larger sample sizes to elucidate the role of CNVs in AMD pathogenesis.

Reproducibility of choroidal thickness measurements in subjects on 3 spectral domain optical coherence tomography machines.

The purpose of this study was to investigate the reproducibility of choroidal thickness measurements in normal subjects on 3 spectral domain optical coherence tomography instruments, namely: Zeiss Cirrus HD-OCT (Carl Zeiss Meditec Inc., Dublin, CA), Heidelberg Spectralis (Heidelberg Engineering, Heidelberg, Germany), and Optovue RTVue (Optovue Inc., Fremont, CA). This cross-sectional non-interventional study was performed in a single institution. Images were obtained in 47 eyes of 47 healthy volunteers which age ranged between 23 and 72 without ocular pathology. All subjects were imaged on the fovea using Cirrus HD 1-line raster, Spectralis enhanced depth imaging, and RTVue retina-cross. The choroid was measured subfoveally and at intervals of 500 µm from the fovea nasally and temporally up to 2500 µm. Paired t test, modified Bland-Altman plot, and Pearson's correlation were used to compare the results. There is no significant difference between the systems for any measurement within 2500 µm either side of the fovea for most points. Inter-observer correlation was strong for RTVue, and moderate in both Cirrus and Spectralis.

Association of potential salivary biomarkers with diabetic retinopathy and its severity in type-2 diabetes mellitus: a proteomic analysis by mass spectrometry.

AIM/HYPOTHESIS: The aim of our study was to characterize the human salivary proteome and determine the changes in protein expression in two different stages of diabetic retinopathy with type-2 diabetes mellitus: (1) with non-proliferative diabetic retinopathy (NPDR) and (2) with proliferative diabetic retinopathy (PDR). Type-2 diabetes mellitus without diabetic retinopathy (XDR) was designated as control. METHOD: In this study, 45 saliva samples were collected (15 samples from XDR control group, 15 samples from NPDR disease group and 15 samples from PDR disease group). Salivary proteins were extracted, reduced, alkylated, trypsin digested and labeled with an isobaric tag for relative and absolute quantitation (iTRAQ) before being analyzed by an Orbitrap fusion tribrid mass spectrometer. Protein annotation, fold change calculation and statistical analysis were interrogated by Proteome Discoverer. Biological pathway analysis was performed by Ingenuity Pathway Analysis. Data are available via ProteomeXchange with identifiers PXD003723-PX003725. RESULTS: A total of 315 proteins were identified from the salivary proteome and 119 proteins were found to be differentially expressed. The differentially expressed proteins from the NPDR disease group and the PDR disease group were assigned to respective canonical pathways indicating increased Liver X receptor/Retinoid X receptor (LXR/RXR) activation, Farnesoid X receptor/Retinoid X receptor (FXR/RXR) activation, acute phase response signaling, sucrose degradation V and regulation of actin-based motility by Rho in the PDR disease group compared to the NPDR disease group. CONCLUSIONS/INTERPRETATION: Progression from non-proliferative to proliferative retinopathy in type-2 diabetic patients is a complex multi-mechanism and systemic process. Furthermore, saliva was shown to be a feasible alternative sample source for diabetic retinopathy biomarkers.

Visual System Homeobox 1 (VSX1) Gene Analysis in Keratoconus: Design of Specific Primers and DNA Amplification Protocols for Accurate Molecular Characterization.

BACKGROUND: Keratoconus is an ocular degeneration characterized by the thinning of corneal stroma that may lead to varying degrees of myopia and visual impairment. Genetic factors have been reported in the pathology of keratoconus where Asians have a higher incidence, earlier onset, and undergo earlier corneal grafts compared to Caucasians. The visual system homeobox 1 (VSX1) gene forms part of a paired-like homeodomain transcription factor which is responsible for ocular development. The gene was marked as a candidate in genetic studies of keratoconus in various populations. Single nucleotide polymorphisms (SNPs) in the VSX1 gene have been reported to be associated with keratoconus. The detection of the SNPs involves DNA amplification of the VSX1 gene followed by genomic sequencing. Thus, the objective of this study aims to establish sensitive and accurate screening protocols for the molecular characterization of VSX1 polymorphisms. METHODS: Keratoconic (n = 74) and control subjects (n = 96) were recruited based on clinical diagnostic tests and selection criteria. DNA extracted from the blood samples was used to genotype VSX1 polymorphisms. In-house designed primers and optimization of PCR conditions were carried out to amplify exons 1 and 3 of the VSX1 gene. PCR conditions including percentage GC content, melting temperatures, and differences in melting temperatures of primers were evaluated to produce sensitive and specific DNA amplifications. RESULTS: Genotyping was successfully carried out in 4 exons of the VSX1 gene. Primer annealing temperatures were observed to be crucial in enhancing PCR sensitivity and specificity. Annealing temperatures were carefully evaluated to produce increased specificity, yet not allowing sensitivity to be compromised. In addition, exon 1 of the VSX1 gene was amplified using 2 different sets of primers to produce 2 smaller amplified products with absence of non-specific bands. DNA amplification of exons 1 and 3 consistently showed single band products which were successfully sequenced to yield reproducible data. CONCLUSIONS: The use of in-house designed primers and optimized PCR conditions allowed sensitive and specific DNA amplifications that produced distinct single bands. The in-house designed primers and DNA amplification protocols established in this study provide an addition to the current repertoire of primers for accurate molecular characterization of VSX1 gene polymorphisms in keratoconus research.

Compressive optic neuropathy due to a large Onodi air cell: A case report and literature review.

BACKGROUND: To report an unusual case of compressive optic neuropathy secondary to a large onodi air cell. METHOD: Case report. RESULTS: A 50 year-old gentlemen presented to the eye clinic with left eye painless loss of vision for one day. Visual acuity was counting finger in the left eye with a positive relative afferent pupillary defect (RAPD). Dilated left fundus examination revealed a pale optic disc. A computed tomography of orbit and brain showed a large left sphenoid sinus with onodi-cell-like projection on the left superior margin of left optic canal impinging on the left optic nerve. He was referred to the otorhinolaryngology team and subsequently underwent left optic nerve decompression. Post-operatively, his left visual acuity improved to 6/60 with reversal of RAPD. CONCLUSION: There are many causes of optic neuropathy and compressive optic neuropathy due to large onodi air cell is uncommon. Acute unilateral loss of vision heralds from a multitude of sinister causes and junior residents should be vigilant that onodi air cell pneumotisation could be one of them.

Effects of ranibizumab on TGF-ß1 and TGF-ß2 production by human Tenon's fibroblasts: An in vitro study.

PURPOSE: Inhibiting exaggerated wound healing responses, which are primarily mediated by human Tenon's fibroblast (HTF) migration and proliferation, has become the major determining factor for a successful trabeculectomy. Antivascular endothelial growth factor (anti-VEGF) has showed promising results as a potential antifibrotic candidate for use concurrently in trabeculectomy. Preliminary cohort studies have revealed improved bleb morphology following trabeculectomy augmented with ranibizumab. However, the effects on HTFs remain unclear. This study was conducted to understand the effects of ranibizumab on transforming growth factor (TGF)-ß1 and transforming growth factor (TGF)-ß2 expression by HTFs. METHODS: The effect of ranibizumab on HTF proliferation and cell viability was determined using 3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. Ranibizumab at concentrations ranging from 0.01 to 0.5 mg/ml were administered for 24, 48, and 72 h in serum and serum-free conditions. Supernatants and cell lysates from samples were assessed for TGF-ß1 and TGF-ß2 mRNA and protein levels using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: At 48 h, 0.5 mg/ml of ranibizumab significantly induced cell death under serum-free culture conditions (p<0.05). Ranibizumab caused a significant reduction in TGF-ß1 mRNA, but not for TGF-ß2. However, the total protein production of TGF-ß1 and TGF-ß2 was unaffected by this anti-VEGF treatment. CONCLUSIONS: Exposure of HTFs to an intravitreal dose of ranibizumab significantly suppresses cell viability in vitro; however, the application seemed unable to affect the ultimate production of TGF-ß. Therefore, we highlighted ranibizumab as a potential antiscarring agent that acts via a different mechanism when used synergistically with another antifibrotic agent. Understanding the mechanism of actions of ranibizumab offers an additional view of a possible new rational therapeutic strategy.

Nasal chondroma presenting as hypertelorism.

PURPOSE: To report a rare case of nasal chondroma presenting as hypertelorism. CASE REPORT: We report a case of a 16-year-old boy with a large calcified mass arising from the posterior nasal cavity presenting as hypertelorism. Surgical excision was done, and the histopathological examination revealed a chondroma. The hypertelorism resolved postoperatively. CONCLUSIONS: Nasal chondroma may also present innocuously as hypertelorism as in this case.

Accutome PachPen handheld ultrasonic pachymeter: intraobserver repeatability and interobserver reproducibility by personnel of different training grades.

To assess the intra-observer repeatability and inter-observer reproducibility of central corneal thickness (CCT) measurements of PachPen (Accutome, Inc., Pennsylvania, USA), a hand-held, portable ultrasonic pachymeter when used by an ophthalmic nurse compared to an ophthalmologist. Ophthalmology Clinic, University of Malaya Medical Center In this prospective study, CCT was measured in 184 eyes of 92 healthy subjects, first by a corneal surgeon experienced in ultrasound pachymetry (Observer 1) followed by an ophthalmic nurse new to the procedure (Observer 2). Nine measurements were obtained from each eye by each observer, independently. Measurements were compared between the observers. Coefficients of repeatability and reproducibility were calculated. The Bland-Altman plot was used to assess agreement between observers. Mean age of the study population was 54.3 ± 15.2 years old and consisted of 43.5% male. Mean CCT as measured by Observers 1 and 2 were 528.3 ± 32.9 and 530.7 ± 33.3 µm, respectively. Observer 1 showed higher repeatability of measurements compared to that of Observer 2 (coefficient of repeatability 3.46 vs. 5.55%). The measurements by both observers showed high correlation (0.96) and good agreement (mean difference -2.4 µm; 95% limits of agreement -21.4, 16.7 µm). Coefficient of reproducibility of measurements between observers was 5.08%. Accutome PachPen hand-held ultrasound pachymeters gives excellent intra-observer repeatability and inter-observer reproducibility by personnel of different training grades.

Phospholipase A2 group v in benign familial fleck retina in a set of triplets.

PURPOSE: To evaluate the association of phospholipase A2, Group V (PLA2G5), with benign familial fleck retina in a consanguineous family with triplets. METHODS: Clinical eye examination, including fundus examination and spectral domain optical coherence tomography, was performed for all the family members. After blood sample collection and DNA extraction, polymerase chain reaction was performed to amplify regions spanning Exons 2, 3, 4, and 5 of PLA2G5. The amplified products were sequenced to observe the presence of any mutations. RESULTS: Fundus examination in two of the triplets revealed discrete yellow-white flecks and both had good vision and absence of night blindness, consistent with benign familial fleck retina. The flecks were hyperautofluorescent. Furthermore, spectral domain optical coherence tomography showed focal thickening of the retinal pigment epithelium because of the presence of these flecks. Molecular investigations showed that PLA2G5 Exons 2, 4, and 5 harbored no misalignments among all family members. However, PLA2G5 Exon 3 showed a p.Gly45Cys mutation for the father and the third triplet who was affected. CONCLUSION: The clinical findings in this family suggest a diagnosis of benign familial fleck retina with excellent prognosis, in which the PLA2G5 gene may play a role.

The trend of retinopathy of prematurity in Malaysia from 1992 to 2001 based on a nationwide blind schools study.

Retinopathy of prematurity (ROP) is one of the most important causes of childhood blindness worldwide. The trend of ROP in Malaysia was unclear because there was no national registry before 2002. The purpose of this study is to analyze ROP students of different ages in the schools for the blind in Malaysia in order to evaluate the trend of ROP from 1992 to 2001. Data were obtained from a previous survey of 24 blind schools. It was found that 78 students or 17.4% were blind/severely visual impaired as a result of ROP. There was a significant surge in the number of ROP students who were born in 1994 when the use of synthetic surfactants was first introduced in Malaysia; otherwise there was no increasing trend in the number of students with ROP. However, the percentage of ROP in total was increasing, which indicates that ROP is becoming a more important cause of childhood blindness in this country.

Refractive outcomes comparison between the Lenstar LS 900® optical biometry and immersion A-scan ultrasound.

To determine the accuracy of intraocular lens (IOL) calculations in eyes undergoing phacoemulsification cataract surgery with IOL implantation using immersion A-scan ultrasound (US) and Lenstar LS 900(®) biometry. In this prospective study, 200 eyes of 200 patients were randomized to undergo either Lenstar LS 900(®) or immersion A-scan US biometry to determine the IOL dioptric power prior to phacoemulsification cataract surgery. Post-operative refractive outcomes of these two groups of patients were compared. The result showed no significant difference between the target spherical equivalent (SE) and the post-operative SE value by the Lenstar LS 900(®) (p value = 0.632) or immersion A-scan US biometry (p value = 0.438) devices. The magnitude of difference between the two biometric devices were not significantly different (p value = 0.868). There was no significant difference in the predicted post-operative refractive outcome between immersion A-scan US biometry and Lenstar LS 900(®). Based on the results, the immersion A-scan US technique is as accurate as Lenstar LS 900(®) in the hands of an experienced operator.

Association between Ocular Pseudoexfoliation and Sensorineural Hearing Loss.

Background. Our study aimed to investigate an association between ocular pseudoexfoliation (PXF) and sensorineural hearing loss (SNHL) and to compare them with age and sex matched controls without pseudoexfoliation. Method. This was a case-control study of 123 patients which included 68 cases with PXF (at least one eye) and 55 controls without pseudoexfoliation. Pure-tone audiometry (PTA) was done for these patients at sound frequencies taken as important for speech comprehension, that is, 250 Hertz (Hz), 500 Hz, 1000 Hz, and 2000 Hz. Results. There were 41 patients with pseudoexfoliation syndrome (PXE) and 27 with pseudoexfoliative glaucoma (PXEG). The majority of patients with hearing loss (60%; n = 51) were PXF patients and the remaining 40% (n = 34) were controls. Below average hearing thresholds were significantly higher in the pseudoexfoliation group compared to the control group (P = 0.01; odds ratio (OR), 3.00; 95% confidence interval (CI), 1.25-7.19). However, there was no significant difference in the mean hearing threshold levels between the three groups (PXE, PXEG, and controls) in either ear (ANOVA, right ear: P = 0.46 and left ear P = 0.36). Conclusion. Our study found an association between PXF and SNHL, confirming that PXF can involve organs in the body other than the eye.