MicroRNA-155 (miR-155) and STAT3 are implicated in uveitis and pathogenic mechanisms of CNS autoimmune diseases. In our study, we used miR-155−/− mice and mice with targeted STAT3 deletion in T cells (CD4-STAT3KO) to investigate roles of miR-155 and STAT3 in the development of experimental autoimmune uveitis (EAU), a mouse model of human uveitis.
We induced EAU in WT, miR-155−/−, or CD4-STAT3KO mice by immunization with interphotoreceptor retinoid-binding protein/complete Freund's adjuvant (IRBP/CFA) or adoptive transfer of T cells. EAU was assessed by funduscopy and histology. RNA expression was analyzed by quantitative PCR (qPCR), while cytokine production was assessed by fluorescence-activated cell sorting (FACS).
We used a combination of genomic and genetic tools to provide the first evidence that STAT3 binds directly to the miR-155 locus and that STAT3 is required for miR-155 expression. Furthermore, STAT3-dependent increase in miR-155 expression in vivo correlated temporally with onset of EAU, and miR-155−/− or CD4-STAT3KO mice did not suffer EAU. CD4+ lymph node cells from IRBP-immunized WT mice transferred EAU to naïve wild-type (WT) and miR-155−/− mice, while miR-155−/− IRBP-specific T cells did not.
Although miR-155 and STAT3 have been implicated in the etiology of multiple sclerosis (MS), uveitis, or rheumatoid arthritis, their exact roles in these diseases are unclear. We show here for the first time to our knowledge that STAT3 regulates miR-155 expression in Th17 cells. We show further that STAT3 and miR-155 form an axis that promotes the expansion of pathogenic Th17 cells that mediate uveitis. Thus, STAT3 and miR-155 may be therapeutic targets for treating uveitis and other Th17-mediated inflammatory disorders.
We show for the first time, to our knowledge, that STAT3 regulates miR-155 expression by Th17 cells and a STAT3/miR-155 axis mediates uveitis by promoting Th17 expansion. Data suggest that therapeutic strategies that combine miR-155 inhibition with blockade of STAT3 signaling may ameliorate Th17-mediated autoimmune disease.
EAU; STAT3; miR-155; uveitis; Th17 cells
To assess the ultrastructure of the epithelial basement membrane using transmission electron microscopy (TEM) in rabbit corneas with and without subepithelial stroma opacity (haze).
Two groups of eight rabbits each were included in this study. Photorefractive keratectomy (PRK) was performed using an excimer laser. The first group had −4.5-diopter (−4.5D) PRK and the second group had −9.0D PRK. Contralateral eyes were unwounded controls. Rabbits were sacrificed at 4 weeks after surgery. Immunohistochemical analysis was performed to detect the myofibroblast marker α-smooth muscle actin (SMA). TEM was performed to analyze the ultrastructure of the epithelial basement membrane and stroma.
At 4 weeks after PRK, α-SMA+ myofibroblasts were present at high density in the subepithelial stroma of rabbit eyes that had −9.0D PRK, along with prominent disorganized extracellular matrix, whereas few myofibroblasts and little disorganized extracellular matrix were noted in eyes that had −4.5D PRK. The epithelial basement membrane was irregular and discontinuous and lacking typical morphology in all corneas at 1 month after −9D PRK compared to corneas at 1 month in the −4.5D PRK group.
The epithelial basement membrane acts as a critical modulator of corneal wound healing. Structural and functional defects in the epithelial basement membrane correlate to both stromal myofibroblast development from precursor cells and continued myofibroblast viability, likely through the modulation of epithelial–stromal interactions mediated by cytokines. Prolonged stromal haze in the cornea is associated with abnormal regeneration of the epithelial basement membrane.
The epithelial basement membrane fails to regenerate normally in corneas that develop opacity (haze) after photorefractive keratectomy. This structural abnormality, and associated lack of barrier function, likely underlies the development of haze generated by myofibroblasts.
basement membrane; corneal epithelium; myofibroblasts; haze; corneal wound healing
While the role of the macular pigment carotenoids in the prevention of age-related macular degeneration has been extensively studied in adults, comparatively little is known about the physiology and function of lutein and zeaxanthin in the developing eye. We therefore developed a protocol using a digital video fundus camera (RetCam) to measure macular pigment optical density (MPOD) and distributions in premature infants and in children.
We used blue light reflectance to image the macular pigment in premature babies at the time of retinopathy of prematurity (ROP) screening and in children aged under 7 years who were undergoing examinations under anesthesia for other reasons. We correlated the MPOD with skin carotenoid levels measured by resonance Raman spectroscopy, serum carotenoids measured by HPLC, and dietary carotenoid intake.
We enrolled 51 infants and children ranging from preterm to age 7 years. MPOD correlated significantly with age (r = 0.36; P = 0.0142), with serum lutein + zeaxanthin (r = 0.44; P = 0.0049) and with skin carotenoid levels (r = 0.42; P = 0.0106), but not with dietary lutein + zeaxanthin intake (r = 0.13; P = 0.50). All premature infants had undetectable macular pigment, and most had unusually low serum and skin carotenoid concentrations.
Our most remarkable finding is the undetectable MPOD in premature infants. This may be due in part to foveal immaturity, but the very low levels of serum and skin carotenoids suggest that these infants are carotenoid insufficient as a consequence of low dietary intake and/or severe oxidative stress. The potential value of carotenoid supplementation in the prevention of ROP and other disorders of prematurity should be a fruitful direction for further investigation.
Little is known about the physiology and function of lutein and zeaxanthin in the developing eye. We therefore developed a protocol using a digital fundus camera (RetCam) to measure macular pigment optical density (MPOD) and distributions in premature infants and in children.
macular pigment; carotenoid; imaging; lutein; zeaxanthin
Glucocorticoids (GCs) effectively reduce retinal edema and induce vascular barrier properties but possess unwanted side effects. Understanding GC induction of barrier properties may lead to more effective and specific therapies. Previous work identified the occludin enhancer element (OEE) as a GC-responsive cis-element in the promoters of multiple junctional genes, including occludin, claudin-5, and cadherin-9. Here, we identify two OEE-binding factors and determine their contribution to GC induction of tight junction (TJ) gene expression and endothelial barrier properties.
OEE-binding factors were isolated from human retinal endothelial cells (HREC) using DNA affinity purification followed by MALDI-TOF MS/MS. Chromatin immunoprecipitation (ChIP) assays determined in situ binding. siRNA was used to evaluate the role of trans-acting factors in transcription of TJ genes in response to GC stimulation. Paracellular permeability was determined by quantifying flux through a cell monolayer, whereas transendothelial electrical resistance (TER) was measured using the ECIS system.
MS/MS analysis of HREC nuclear extracts identified the heterodimer of transcription factors p54/NONO (p54) and polypyrimidine tract-binding protein-associated splicing factor (PSF) as OEE-binding factors, which was confirmed by ChIP assay from GC-treated endothelial cells and rat retina. siRNA knockdown of p54 demonstrated that this factor is necessary for GC induction of occludin and claudin-5 expression. Further, p54 knockdown ablated the pro-barrier effects of GC treatment.
p54 is essential for GC-mediated expression of occludin, claudin-5, and barrier induction, and the p54/PSF heterodimer may contribute to normal blood-retinal barrier (BRB) induction in vivo. Understanding the mechanism of GC induction of BRB properties may provide novel therapies for macular edema.
We have identified the p54 NONO/PSF heterodimer as a binding partner of the occludin enhancer that is necessary for glucocorticoid induction of barrier properties.
tight junction; blood-retinal barrier; enhancer; gene expression
StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed.
Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery.
Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment.
In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.
Subretinal delivery of an EIAV based lentiviral vector in the macaque eye leads to efficient gene expression in the photoreceptor cells leading to the development of a gene therapy program to deliver the normal functional copy of ABCA4 in the photoreceptor cells of individuals with Stargardt disease.
lentivirus; gene therapy; photoreceptors
Protein acetylation is an essential mechanism in regulating transcriptional and inflammatory events. Studies have shown that nonselective histone deacetylase (HDAC) inhibitors can protect the retina from ischemic injury in rats. However, the role of specific HDAC isoforms in retinal degenerative processes remains obscure. The purpose of this study was to investigate the role of HDAC2 isoform in a mouse model of ischemic retinal injury.
Localization of HDAC2 in mice retinas was evaluated by immunohistochemical analyses. To investigate whether selective reduction in HDAC2 activity can protect the retina from ischemic injury, Hdac2+/− mice were utilized. Electroretinographic (ERG) and morphometric analyses were used to assess retinal function and morphology.
Our results demonstrated that HDAC2 is primarily localized in nuclei in inner nuclear and retinal ganglion cell layers, and HDAC2 activity accounted for approximately 35% of the total activities of HDAC1, 2, 3, and 6 in the retina. In wild-type mice, ERG a- and b-waves from ischemic eyes were significantly reduced when compared to pre-ischemia baseline values. Morphometric examination of these eyes revealed significant degeneration of inner retinal layers. In Hdac2+/− mice, ERG a- and b-waves from ischemic eyes were significantly greater than those measured in ischemic eyes from wild-type mice. Morphologic measurements demonstrated that Hdac2+/− mice exhibit significantly less retinal degeneration than wild-type mice.
This study demonstrated that suppressing HDAC2 expression can effectively reduce ischemic retinal injury. Our results support the idea that the development of selective HDAC2 inhibitors may provide an efficacious treatment for ischemic retinal injury.
This study displayed the localization of HDAC2 in the inner retina and demonstrated that HDAC2 appears to be a key isoform involved in ischemic retinal degeneration. Furthermore, suppressing HDAC2 expression can effectively reduce ischemic retinal injury.
HDAC2; ischemia; retinal degeneration; neuroprotection
Th17 cells are believed to be the primary effector cells in the pathogenesis of dry eye disease (DED). However, the mechanisms by which Th17 cells migrate from the lymphoid tissues to the ocular surface are unknown. The purpose of this study was to investigate the role of the C–C chemokine receptor 6/C–C chemokine ligand 20 (CCR6/CCL20) chemokine axis in mediating Th17 cell migration in DED.
DED was induced by housing C57BL/6 mice in a low-humidity environment supplemented with scopolamine treatment. Th17 cell expression of CCR6 was evaluated using flow cytometry and ocular surface expression of CCL20 was measured using PCR and ELISA assays. CCL20 neutralizing antibody was administered subconjunctivally to DED mice and disease severity, including the frequency of conjunctival Th17 cells, was evaluated.
CCR6 is preferentially expressed by Th17 cells in both normal and DED mice and DED significantly upregulates ocular surface expression of CCL20. Disruption of CCR6/CCL20 binding with CCL20 neutralizing antibody decreases T-cell migration in vitro and reduces Th17 cell infiltration of the conjunctiva when administered in vivo, significantly improving clinical signs of DED. These changes were accompanied by a decrease in ocular surface inflammatory cytokine levels and corneal CD11b+ cell frequencies. Treatment also significantly reduced the generation of Th17 cells.
Local neutralization of CCL20 decreases Th17 cell infiltration of the ocular surface in DED, leading to improvement in clinical signs of disease. This suggests that CCR6/CCL20 interactions direct Th17 cell migration in DED and that disruption of this axis may be a novel therapeutic approach to this condition.
This study investigates the role of the CCR6/CCL20 axis in directing Th17 cell migration in dry eye. Disruption of CCR6/CCL20 binding prevents Th17 cell migration to the ocular surface and improves disease severity.
dry eye; Th17 cell; cell migration; CCL20; CCR6
To investigate if topical treatment of neuroprotectin D1 (NPD1) increases regeneration of functional nerves after lamellar keratectomy.
An 8-mm stromal dissection was performed in the left eye of each rabbit. The rabbits were treated with NPD1, pigment epithelial-derived factor (PEDF) in combination with docosahexaenoic acid (DHA) or vehicle for 6 weeks, and corneas were obtained at 8 weeks. After fixation, corneal wholemounts were stained with mouse monoclonal anti-βIII-tubulin antibody and double stained with chicken anti-calcitonin gene-related peptide (CGRP) antibody. Corneal sensitivity and tear secretion were measured using the Cochet-Bonnet esthesiometer and the Schirmer's test, respectively. Additional rabbits were treated with NPD1, PEDF+DHA, or vehicle, and corneal sections were stained with a rat monoclonal anti-neutrophil antibody. Cultures of trigeminal ganglia from 5-day-old mice were treated with NPD1, PEDF+DHA, lipoxin A4 (LXA4), 12- or 15-hydroxyeicosatetraenoic acid (12[S] or 15[S]-HETE), and nerve growth factor (NGF) as positive control.
NPD1 increased subepithelial corneal nerve area three times compared with vehicle-treated rabbits. The effect was similar to PEDF+DHA–treated animals. There was recovery of CGRP-positive neurons and an increase in corneal sensitivity and tear secretion in NPD1-treated animals. NPD1 decreased neutrophil infiltration after 2 and 4 days of treatment. In the in vitro cultures, NPD1 and PEDF+DHA induced a 3-fold increase in neurite outgrowth compared with cultures without supplementation. Treatments with LXA4, 12(S)-, and 15(S)- HETE did not stimulate neurite outgrowth.
NPD1 has anti-inflammatory and nerve regenerative properties. This study demonstrates that NPD1 may offer an effective treatment for neurotrophic corneas.
We show, for the first time, that the docosanoid neuroprotectin D1 (NPD1) is able to regenerate corneal nerves after damage from lamellar keratectomy in an animal model and reduce polymorphonuclear neutrophil infiltration induced by injury.
neuroregeneration; corneal sensitivity; pigment epithelial-derived factor; neuroprotectin D1; dry eye
To determine whether choroidal imaging is feasible in preterm and term infants using an 840-nm portable spectral domain optical coherence tomography (SD-OCT) system without the use of enhanced-depth imaging techniques and to assess choroidal development by comparing choroidal thickness of preterm infants, term infants, and adults.
SD-OCT images were obtained from 86 preterm infants, 59 term infants, and nine adults using a portable SD-OCT system plus nine adults using a tabletop system. An unprocessed image across the macula from one randomly selected eye of each participant was selected for determination of whether the choroidal-scleral junction (CSJ) could be visualized and for measurement of choroidal thickness.
Subfoveal CSJ was visualized in 96% of young-preterm infants (imaged from 30–36 weeks postmenstrual age [PMA]); 78% of term-aged preterm infants (imaged from 37–42 weeks PMA); 49% of term infants; and 39% of adult subjects. Racial pigmentation did not affect CSJ visibility in young-preterm infants (P = 0.57). Subfoveal choroidal thickness (SFCT) in young-preterm infants, term-aged preterm infants, term infants, and adults was 176 ± 53 μm, 289 ± 92 μm, 329 ± 66 μm, and 258 ± 66 μm, respectively, and these were all statistically significantly different from one another except term-aged preterms to adults.
Infant choroid can be imaged with a portable SD-OCT system without enhanced depth imaging. Melanin in the RPE and choroid does not hinder outer choroidal imaging in young-preterm infants without advanced retinopathy of prematurity (ROP). In preterm infants, choroidal thickness increased with age but was thinner when compared to term infants suggesting delayed development due to ROP.
The choroid in preterm and term infants can be imaged with a portable 840-nm SD-OCT system. This finding may be related to less choroidal pigment at this early time of life. Choroidal imaging revealed delayed choroidal development in subjects born prematurely compared with term infants.
choroid; retinopathy of prematurity; ocular development; optical coherence tomography
Retinitis pigmentosa (RP) is a highly heterogeneous genetic disease; therefore, an accurate molecular diagnosis is essential for appropriate disease treatment and family planning. The prevalence of RP in China had been reported at 1 in 3800, resulting in an estimated total of 340,000 Chinese RP patients. However, genetic studies of Chinese RP patients have been very limited. To date, no comprehensive molecular diagnosis has been done for Chinese RP patients. With the emergence of next-generation sequencing (NGS), comprehensive molecular diagnosis of RP is now within reach. The purpose of this study was to perform the first NGS-based comprehensive molecular diagnosis for Chinese RP patients.
Thirty-one well-characterized autosomal recessive RP (arRP) families were recruited. For each family, the DNA sample from one affected member was sequenced using our custom capture panel, which includes 163 retinal disease genes. Variants were called, filtered, and annotated by our in-house automatic pipeline.
Twelve arRP families were successfully molecular diagnosed, achieving a diagnostic rate of approximately 40%. Interestingly, approximately 63% of the pathogenic mutations we identified are novel, which is higher than that observed in a similar study on European descent (45%). Moreover, the clinical diagnoses of two families were refined based on the pathogenic mutations identified in the patients.
We conclude that comprehensive molecular diagnosis can be vital for an accurate clinical diagnosis of RP. Applying this tool on patients from different ethnic groups is essential for enhancing our knowledge of the global spectrum of RP disease-causing mutations.
Next-generation sequencing–based comprehensive molecular diagnosis of 31 families with autosomal recessive retinitis pigmentosa (arRP) was performed. This is the first such study of a Chinese arRP patient cohort, revealing a total of 10 novel putatively pathogenic mutations.
retinitis pigmentosa; next-generation sequencing; molecular diagnosis; Chinese population
To perform a Bayesian analysis of the Mycotic Ulcer Treatment Trial I (MUTT I) using expert opinion as a prior belief.
MUTT I was a randomized clinical trial comparing topical natamycin or voriconazole for treating filamentous fungal keratitis. A questionnaire elicited expert opinion on the best treatment of fungal keratitis before MUTT I results were available. A Bayesian analysis was performed using the questionnaire data as a prior belief and the MUTT I primary outcome (3-month visual acuity) by frequentist analysis as a likelihood.
Corneal experts had a 41.1% prior belief that natamycin improved 3-month visual acuity compared with voriconazole. The Bayesian analysis found a 98.4% belief for natamycin treatment compared with voriconazole treatment for filamentous cases as a group (mean improvement 1.1 Snellen lines, 95% credible interval 0.1–2.1). The Bayesian analysis estimated a smaller treatment effect than the MUTT I frequentist analysis result of 1.8-line improvement with natamycin versus voriconazole (95% confidence interval 0.5–3.0, P = 0.006). For Fusarium cases, the posterior demonstrated a 99.7% belief for natamycin treatment, whereas non-Fusarium cases had a 57.3% belief.
The Bayesian analysis suggests that natamycin is superior to voriconazole when filamentous cases are analyzed as a group. Subgroup analysis of Fusarium cases found improvement with natamycin compared with voriconazole, whereas there was almost no difference between treatments for non-Fusarium cases. These results were consistent with, though smaller in effect size than, the MUTT I primary outcome by frequentist analysis. The accordance between analyses further validates the trial results. (ClinicalTrials.gov number, NCT00996736.)
We elicited the opinions of corneal specialists on treating filamentous fungal keratitis, to perform a Bayesian analysis of the Mycotic Ulcer Treatment Trial I. The Bayesian analysis result was consistent with, but suggested a smaller treatment effect than, the frequentist result.
fungal keratitis; corneal ulceration; clinical trial; Bayesian; statistics
We evaluated the ability of baseline and longitudinal estimates of retinal ganglion cell (RGC) counts in predicting progression in eyes suspected of having glaucoma.
The study included 288 glaucoma suspect eyes of 288 patients followed for an average of 3.8 ± 1.0 years. Participants had normal standard automated perimetry (SAP) at baseline. Retinal nerve fiber layer thickness assessment was performed with optical coherence tomography (OCT). Progression was defined as development of repeatable abnormal SAP or glaucomatous progressive optic disc changes. Estimates of RGC counts were obtained by combining data from SAP and OCT according to a previously described method. Joint longitudinal survival models were used to evaluate the ability of baseline and rates of change in estimated RGC counts for predicting progression over time, adjusting for confounding variables.
A total of 48 eyes (17%) showed progression during follow-up. The mean rate of change in estimated RGC counts was −18,987 cells/y in progressors versus −8,808 cells/y for nonprogressors (P < 0.001). Baseline RGC counts and slopes of RGC loss were significantly predictive of progression, with HRs of 1.56 per 100,000 cells lower (95% confidence interval [CI], 1.18–2.08; P = 0.002) and 2.68 per 10,000 cells/y faster loss (95% CI, 1.22–5.90; P = 0.014), respectively. The longitudinal model including estimates of RGC counts performed significantly better than models including only structural or functional indexes separately.
Baseline and longitudinal estimates of RGC counts may be helpful in predicting progression and performed significantly better than conventional approaches for risk stratification of glaucoma suspects.
This study demonstrated that baseline and longitudinal estimates of retinal ganglion cell counts based on combined structure and function tests may be helpful in predicting progression, and performed significantly better than conventional approaches for risk stratification of glaucoma suspects.
glaucoma; visual field; optical coherence tomography; optic nerve head; intraocular pressure
To evaluate the intersession repeatability of retinal thickness measurements in patients with diabetic macular edema (DME) using the Heidelberg Spectralis optical coherence tomography (OCT) algorithm and a publicly available, three-dimensional graph search-based multilayer OCT segmentation algorithm, the Iowa Reference Algorithm.
Thirty eyes from 21 patients diagnosed with clinically significant DME were included and underwent consecutive, registered macula-centered spectral-domain optical coherence scans (Heidelberg Spectralis). The OCT scans were segmented into separate surfaces, and the average thickness between internal limiting membrane and outer retinal pigment epithelium complex surfaces was determined using the Iowa Reference Algorithm. Variability between paired scans was analyzed and compared with the retinal thickness obtained from the manufacturer-supplied Spectralis software.
The coefficient of repeatability (variation) for central macular thickness using the Iowa Reference Algorithm was 5.26 μm (0.62% [95% confidence interval (CI), 0.43–0.71]), while for the Spectralis algorithm this was 6.84 μm (0.81% [95% CI, 0.55–0.92]). When the central 3 mm was analyzed, the coefficient of repeatability (variation) was 2.46 μm (0.31% [95% CI, 0.23–0.38]) for the Iowa Reference Algorithm and 4.23 μm (0.53% [95% CI, 0.39–0.65]) for the Spectralis software.
The Iowa Reference Algorithm and the Spectralis software provide excellent reproducibility between serial scans in patients with clinically significant DME. The publicly available Iowa Reference Algorithm may have lower between-measurement variation than the manufacturer-supplied Spectralis software for the central 3 mm subfield. These findings have significant implications for the management of patients with DME.
Intersession reproducibility of DME imaged by Heidelberg Spectralis OCT was excellent using two different segmentation algorithms. Coefficient of repeatability of central macular thickness using the Heidelberg supplied software was 6.84 μm and using the Iowa Reference Algorithm was 5.26 μm.
OCT; diabetic retinopathy; image analysis; macular edema; retina
The aim of this study was to explore how different statistical methods may lead to inconsistent inferences about the association between structure and function in glaucoma.
Two datasets from published studies were selected for their illustrative value. The first consisted of measurements of neuroretinal rim area in the superior-temporal sector paired with the corresponding visual field sensitivity. The second consisted of measurements of average retinal nerve fiber layer thickness over all sectors paired with the corresponding visual field sensitivity. Statistical methods included linear and segmented regression, and a nonparametric local-linear fit known as loess. The analyses were repeated with all measurements expressed as percent of mean normal.
Slopes from linear fits to the data changed by a factor of 10 depending on the linear regression method applied. Inferences about whether structural abnormality precedes functional abnormality varied with the statistical design and the units of measure used.
The apparent association between structure and function in glaucoma, and consequent interpretation, varies with the statistical method and units of measure. Awareness of the limitations of any statistical analysis is necessary to avoid finding spurious results that ultimately may lead to inadequate clinical recommendations.
Studies comparing structural and functional damage from glaucoma often use statistical methods such as linear regression, which has limitations that can lead to inadequate clinical recommendations. These limitations are analyzed, using examples from the literature.
ordinary least-squares regression; segmented regression; loess; structure-function relationships
To develop new therapies against ocular neovascularization (NV), we tested the effect of peroxisome proliferator-activated receptor-β/δ (PPAR-β/δ) agonism and antagonism on angiogenic behaviors and in human retinal microvascular endothelial cells (HRMEC) and on preretinal NV in rat oxygen-induced retinopathy (OIR).
HRMECs were treated with the PPAR-β/δ agonist GW0742 and the antagonist GSK0660. Messenger RNA levels of a PPAR-β/δ target gene, angiopoietin-like-4 (angptl4) were assayed by qRT-PCR. HRMEC proliferation and tube formation were assayed according to standard protocols. OIR was induced in newborn rats by exposing them to alternating 24-hour episodes of 50% and 10% oxygen for 14 days. OIR rats were treated with GW0742 or GSK0660. Angptl4 protein levels were assessed by ELISA and preretinal NV was quantified by adenosine diphosphatase staining.
GW0742 significantly increased angptl4 mRNA, and GSK0660 significantly decreased angptl4 mRNA. GW0742 had no effect on HRMEC proliferation, but caused a significant and dose-responsive increase in tube formation. GSK0660 significantly reduced serum-induced HRMEC proliferation and tube formation in a dose-dependent manner. Intravitreal injection of GW0742 significantly increased total retinal Angptl4 protein, but intravitreal injection of GSK0660 had no effect. Intravitreal injection of GW0742 significantly increased retinal NV, as did GW0742 administered by oral gavage. Conversely, both intravitreal injection and intraperitoneal injection of GSK0660 significantly reduced retinal NV.
PPAR-β/δ activation exacerbates, and its inhibition reduces, preretinal NV. PPAR-β/δ may regulate preretinal NV through a prodifferentiation/maturation mechanism that depends on Angptl4. Pharmacologic inhibition of PPAR-β/δ may provide a rational basis for therapeutic targeting of ocular NV.
The goal of this study was to determine the effect of pharmacologic manipulation of PPAR-β/δ on retinal angiogenesis. We found that inhibition of PPAR-β/δ reduces neovascularization in the rat model of oxygen-induced retinopathy.
retinopathy of prematurity; nuclear transcription factor; angiogenesis; vascular endothelial growth factor; peroxisome proliferator-activated receptor
Meibomian gland dysfunction (MGD) is a primary cause of dry eye disease. One of the risk factors for MGD is exposure to 13-cis retinoic acid (13-cis RA), a metabolite of vitamin A. However, the mechanism is not well understood. We hypothesize that 13-cis RA inhibits cell proliferation, promotes cell death, alters gene and protein expressions, and attenuates cell survival pathways in human meibomian gland epithelial cells.
To test our hypotheses, immortalized human meibomian gland epithelial cells were cultured with or without 13-cis RA for varying doses and time. Cell proliferation, cell death, gene expression, and proteins involved in proliferation/survival and inflammation were evaluated.
We found that 13-cis RA inhibited cell proliferation, induced cell death, and significantly altered the expression of 6726 genes, including those involved in cell proliferation, cell death, differentiation, keratinization, and inflammation, in human meibomian gland epithelial cells. Further, 13-cis RA also reduced the phosphorylation of Akt and increased the generation of interleukin-1β and matrix metallopeptidase 9.
Exposure to 13-cis RA inhibits cell proliferation, increases cell death, alters gene expression, changes signaling pathways, and promotes inflammatory mediator and protease expression in meibomian gland epithelial cells. These effects may be responsible, at least in part, for the 13-cis RA–related induction of MGD.
In this study, we address the mechanism of meibomian gland dysfunction induced by 13-cis retinoic acid (RA) in vitro. We found that 13-cis RA alters meibomian gland epithelial cell gene expression, reduces cell survival mediators, inhibits proliferation, and induces meibocyte cell death.
retinoic acid; meibomian gland dysfunction; dry eye disease
To correlate retinal function and visual sensitivity with retinal morphology revealed by ultrahigh-resolution imaging with adaptive optics–optical coherence tomography (AO-OCT), on patients with geographic atrophy.
Five eyes from five subjects were tested (four with geographic atrophy [66.3 ± 6.4 years, mean ± 1 SD] and one normal [61 years]). Photopic and scotopic multifocal electroretinograms (mfERGs) were recorded. Visual fields were assessed with microperimetry (mP) combined with a scanning laser ophthalmoscope for high-resolution confocal retinal fundus imaging. The eye tracker of the microperimeter identified the preferred retinal locus that was then used as a reference for precise targeting of areas for advanced retinal imaging. Images were obtained with purpose-built, in-house, ultrahigh resolution AO-OCT. Fundus autofluorescence (FAF) and color fundus (CF) photographs were also acquired.
The AO-OCT imaging provided detailed cross-sectional structural representation of the retina. Up to 12 retinal layers were identified in the normal subject while many severe retinal abnormalities (i.e., calcified drusen, drusenoid pigment epithelium detachment, outer retinal tubulation) were identified in the retinae of the GA patients. The functional tests showed preservation of sensitivities, although somewhat compromised, at the border of the GA.
The images provided here advance our knowledge of the morphology of retinal layers in GA patients. While there was a strong correlation between altered retinal structure and reduction in visual function, there were a number of examples in which the photoreceptor inner/outer segment (IS/OS) junctions lost reflectivity at the margins of GA, while visual function was still demonstrated. This was shown to be due to changes in photoreceptor orientation near the GA border.
Patients with geographic atrophy were tested with a battery of functional and structural tests including mfERGs and AO-OCT. There were a number of examples in which the photoreceptor IS/OS junction lost reflectivity at the margins of GA, but visual function was still demonstrated.
geographic atrophy; adaptive-optics OCT; multifocal ERG; scotopic mfERG; microperimetry
We characterized electrically elicited visual evoked potentials (eVEPs) in Argus II retinal implant wearers.
eVEPs were recorded in four subjects, and analyzed by determining amplitude and latency of the first two positive peaks (P1 and P2). Subjects provided subjective feedback by rating the brightness and size of the phosphenes. We established eVEP input–output relationships, eVEP variability between and within subjects, the effect of stimulating different areas of the retina, and the maximal pulse rate to record eVEPs reliably.
eVEP waveforms had low signal-to-noise ratios, requiring long recording times and substantial signal processing. Waveforms varied between subjects, but showed good reproducibility and consistent parameter dependence within subjects. P2 amplitude overall was the most robust outcome measure and proved an accurate indicator of subjective threshold. Peak latencies showed small within-subject variability, yet their correlation with stimulus level and subjective rating were more variable than that of peak amplitudes. Pulse rates of up to 2/3 Hz resulted in reliable eVEP recordings. Perceived phosphene brightness declined over time, as reflected in P1 amplitude, but not in P2 amplitude or peak latencies. Stimulating-electrode location significantly affected P1 and P2 amplitude and latency, but not subjective percepts.
While recording times and signal processing are more demanding than for standard visually evoked potential (VEP) recordings, the eVEP has proven to be a reliable tool to verify retinal implant functionality. eVEPs correlated with various stimulus parameters and with perceptual ratings. In view of these findings, eVEPs may become an important tool in functional investigations of retinal prostheses. (ClinicalTrials.gov number NCT00407602.)
We characterized electrically elicited visual evoked potentials (eVEPs) in Argus II retinal implant wearers. eVEPs were correlated significantly with stimulus level and subjective percept. We conclude that eVEPs may become an important tool for intraoperative monitoring and rehabilitation purposes.
retinal implant; visual evoked potential; visual prosthesis; psychophysics
Increase in reactive oxygen species (ROS) is one of the major retinal metabolic abnormalities associated with the development of diabetic retinopathy. NF-E2–related factor 2 (Nrf2), a redox sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch like-ECH-associated protein 1 (Keap1), and moves to the nucleus to regulate the transcription of antioxidant genes including the catalytic subunit of glutamylcysteine ligase (GCLC), a rate-limiting reduced glutathione (GSH) biosynthesis enzyme. Our aim is to understand the role of Nrf2-Keap1-GCLC in the development of diabetic retinopathy.
Effect of diabetes on Nrf2-Keap1-GCLC pathway, and subcellular localization of Nrf2 and its binding with Keap1 was investigated in the retina of streptozotocin-induced diabetic rats. The binding of Nrf2 at GCLC was quantified by chromatin immunoprecipitation technique. The results were confirmed in isolated retinal endothelial cells, and also in the retina from human donors with diabetic retinopathy.
Diabetes increased retinal Nrf2 and its binding with Keap1, but decreased DNA-binding activity of Nrf2 and also its binding at the promoter region of GCLC. Similar impairments in Nrf2-Keap1-GCLC were observed in the endothelial cells exposed to high glucose and in the retina from donors with diabetic retinopathy. In retinal endothelial cells, glucose-induced impairments in Nrf2-GCLC were prevented by Nrf2 inducer tBHQ and also by Keap1-siRNA.
Due to increased binding of Nrf2 with Keap1, its translocation to the nucleus is compromised contributing to the decreased GSH levels. Thus, regulation of Nrf2-Keap1 by pharmacological or molecular means could serve as a potential adjunct therapy to combat oxidative stress and inhibit the development of diabetic retinopathy.
Diabetes increases retinal Nrf2 levels, but decreases its DNA binding activity. Due to increased binding of Nrf2 with its inhibitor, the recruitment of Nrf2 at the promoter of GCLC, a rate-limiting enzyme in GSH biosynthesis, is decreased, resulting in subnormal antioxidant defense system.
antioxidant defense; diabetic retinopathy; Nrf2
To qualitatively classify and compare types of iris insertion among American Caucasians, American Chinese, and mainland Chinese.
Prospective multicenter cross-sectional study. Sex- and age-matched Caucasian, American Chinese, and mainland Chinese cohorts were enrolled. Ultrasound biomicroscopy (UBM) images of the anterior chamber were acquired. Qualitative classification of iris insertion into basal, middle, and apical categories was performed. Proportions of each type of insertion were compared among racial groups. Multivariate logistic regression was carried out adjusting for potential confounders.
Data from 117, 129, and 112 subjects were available for American Caucasian, American Chinese, and mainland Chinese subjects, respectively. The most common type of iris insertion in the superior quadrant was basal insertion in both ethnic Chinese and Caucasians. In the inferior, nasal, and temporal quadrants, Chinese showed significantly higher proportions of nonbasal insertion (P = 0.048, P < 0.0001, P < 0.0001, respectively). After adjusting for confounders, nonbasal insertion was significantly associated with Chinese ethnicity in nasal and temporal quadrants (nasal, OR: 3.1, temporal, OR: 4.8). Increasing proportions of nonbasal insertion were found with advancing age in both Chinese and Caucasians.
Chinese have significantly higher proportions of nonbasal iris insertion in the nasal and temporal quadrants when compared with Caucasians, even after adjusting for ASOCT-measured anterior segment biometry and iris characteristics. Longitudinal studies in patients who have PAC/PACG are needed to fully elucidate the relationship between iris insertion and angle closure development.
Chinese have significantly higher proportions of nonbasal iris insertions in the inferior, nasal, and temporal quadrants when compared to Caucasians, after adjusting for ASOCT measured anterior chamber biometry and iris profile.
UBM; interethnic comparison; iris insertion; angle closure; PACG
To determine the relationships among equivalent intrinsic noise (Neq), sampling efficiency, and contrast sensitivity (CS) in patients with retinitis pigmentosa (RP), where Neq is an estimate of the amount of noise within the visual pathway and sampling efficiency represents the subject's ability to use stimulus information optimally.
Participants included 10 patients with RP aged 10 to 54 years, who had visual acuities of 20/40 or better, and 10 visually normal control subjects aged 22 to 65 years. CS was measured for 2-cycles-per-degree Gabor patch targets presented in the absence of noise (CS0) and in five levels of noise spectral density. Data were fit with a standard linear amplifier model, which provided estimates of Neq and sampling efficiency.
CS0 for the patients ranged from normal to as much as a factor of 3 below the lower limit of normal. All 10 patients had abnormally high Neq, including two patients with normal CS0. In comparison, only two patients had lower-than-normal sampling efficiency, and these two patients also had below-normal CS0. Log CS0 for the patients was correlated significantly with log Neq (r = −0.80, P < 0.05), but not with log efficiency (r = 0.54, P = 0.11).
Low CS was associated with elevated intrinsic noise in this group of RP patients, but even patients with normal CS had elevated noise levels. The results suggest that CS measurement in both the presence and absence of luminance noise can provide important information about visual dysfunction in RP patients.
This study demonstrated that patients with retinitis pigmentosa have abnormally high levels of equivalent intrinsic noise, which are correlated significantly with their contrast sensitivity losses.
visual noise; contrast sensitivity; retinitis pigmentosa
Inflammation and immune cells regulate choroidal neovascularization (CNV) and could become therapeutic targets in age-related macular degeneration (AMD). Lymphangiogenesis is a key component of various inflammatory diseases. Whether lymphangiogenesis and lymph node–mediated immunity are involved in the pathogenesis of AMD is not understood.
To investigate lymphangiogenesis in CNV, we generated CNV in animals by laser and studied surgically removed CNV membranes from uveitis and AMD patients. Immunohistochemistry was performed with lymphatic vessel endothelial hyaluronate receptor 1 (LYVE-1) and podoplanin antibodies. VEGF-C and VEGFR-3 expressions were examined with immunohistochemistry and Western blotting. To examine the role of lymph node in CNV, we lasered lymphotoxin alpha–deficient mice (LTα−/−) and measured the CNV volume.
Immunohistochemistry showed that LYVE-1(+) macrophages infiltrated in acutely induced CNV, although lymphatic tubes did not form. CNV membranes from patients did not show LYVE-1(+)podoplanin(+) vessels, suggesting the lack of lymphangiogenesis in AMD and uveitis. Western blots and immunostaining revealed VEGF-C and VEGFR-3 expression in CNV lesions, mainly in macrophages and angiogenic endothelial cells. Using fluorescent microsphere tracers, we show a path for cellular migration from the eye to the cervical lymph nodes (LNs) during CNV. However, CNV injury did not cause LN swelling. CNV volume did not differ between wild-type and LN-deficient mice, suggesting that LN is not a key component of early CNV formation.
Laser-induced CNV is not primarily dependent on acquired immunity, nor does the fundus injury affect peripheral LNs. Our results reveal a previously unknown cellular connection between the ocular fundus and the cervical LNs. This connection that in function resembles lymphatics is actively utilized in CNV.
The role of lymphatics in the posterior chamber has been controversial. This work addresses the potential role of lymphangiogenesis in choroidal neovascularization.
macrophage; LYVE-1; VEGFR-3; VEGF-C; AMD; uveitis
To analyze the relationship among macular ganglion cell complex (GCC) thickness, peripapillary nerve fiber layer (NFL) thickness, and visual field (VF) defects in patients with glaucoma.
A Fourier-domain optical coherence tomography (FD-OCT) system was used to map the macula and peripapillary regions of the retina in 56 eyes of 38 patients with perimetric glaucoma. The macular GCC and peripapillary NFL thicknesses were mapped and standard automated perimetry (SAP) was performed. Loss of GCC and NFL were correlated with the VF map on both a point-by-point and regional basis.
Correlation between GCC thickness and peripapillary NFL thickness produced a detailed correspondence map that demonstrates the arcuate course of the NFL in the macula. Corresponding regions within the GCC, NFL, and VF maps demonstrate significant correlation, once parafoveal retinal ganglion cell (RGC) displacement is taken into account.
There are significant point-specific and regional correlations between GCC loss, NFL loss, and deficits on SAP. Using these different data sources together may improve our understanding of glaucomatous damage and aid in the management of patients with glaucoma.
There is significant region-specific correlation between macular ganglion cell complex thickness and two commonly utilized measures of glaucomatous damage, peripapillary nerve fiber layer thickness and visual field deficits. These parameters may be complementary in managing patients with glaucoma.
optical coherence tomography; visual field; retinal nerve fiber layer; ganglion cell complex
Mutations in SLC4A11, a member of the SLC4 superfamily of bicarbonate transporters, give rise to corneal endothelial cell dystrophies. SLC4A11 is a putative Na+ borate and Na+:OH− transporter. Therefore we ask whether SLC4A11 in corneal endothelium transports borate (B[OH]4−), bicarbonate (HCO3−), or hydroxyl (OH−) anions coupled to Na+.
SLC4A11 expression in cultured primary bovine corneal endothelial cells (BCECs) was determined by semiquantitative PCR, SDS-PAGE/Western blotting, and immunofluorescence staining. Ion transport function was examined by measuring intracellular pH (pHi) or Na+ ([Na+]i) in response to Ringer solutions with/without B(OH)4− or HCO3− after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11.
SLC4A11 is localized to the basolateral membrane in BCEC. B(OH)4− (2.5–10 mM) in bicarbonate-free Ringer induced a rapid small acidification (0.01 pH unit) followed by alkalinization (0.05–0.1 pH unit), consistent with diffusion of boric acid into the cell followed by B(OH)4−. However, the rate of B(OH)4−-induced pHi change was unaffected by overexpression of SLC4A11. B(OH)4− did not induce significant changes in resting [Na+i] or the amplitude and rate of acidification caused by Na+ removal. siRNA-mediated knockdown of SLC4A11 (∼70%) did not alter pHi responses to CO2/HCO3−-rich Ringer, Na+-free induced acidification, or the rate of Na+ influx in the presence of bicarbonate. However, in the absence of bicarbonate, siSLC4A11 knockdown significantly decreased the rate (43%) and amplitude (48%) of acidification due to Na+ removal and recovery (53%) upon add-back. Additionally, the rate of acid recovery following NH4+ prepulse was decreased significantly (27%) by SLC4A11 silencing.
In corneal endothelium, SLC4A11 displays robust Na+-coupled OH− transport, but does not transport B(OH)4− or HCO3−.
SLC4A11 mutations cause corneal endothelial dystrophies, although the function of this putative borate transport protein is unknown. Here we show that SLC4A11 is a Na+-linked pHi regulator that does not transport bicarbonate or borate.
SLC4A11; intracellular pH; intracellular Na+; bicarbonate; borate transport
We investigated the in vitro response of Acanthamoeba trophozoites to electric fields (EFs).
Acanthamoeba castellanii were exposed to varying strengths of an EF. During EF exposure, cell migration was monitored using an inverted microscope equipped with a CCD camera and the SimplePCI 5.3 imaging system to capture time-lapse images. The migration of A. castellanii trophozoites was analyzed and quantified with ImageJ software. For analysis of cell migration in a three-dimensional culture system, Acanthamoeba trophozoites were cultured in agar, exposed to an EF, digitally video recorded, and analyzed at various Z focal planes.
Acanthamoeba trophozoites move at random in the absence of an EF, but move directionally in response to an EF. Directedness in the absence of an EF is 0.08 ± 0.01, while in 1200 mV/mm EF, directedness is significantly higher at −0.65 ± 0.01 (P < 0.001). We find that the trophozoite migration response is voltage-dependent, with higher directionality with higher voltage application. Acanthamoeba move directionally in a three-dimensional (3D) agar system as well when exposed to an EF.
Acanthamoeba trophozoites move directionally in response to an EF in a two-dimensional and 3D culture system. Acanthamoeba trophozoite migration is also voltage-dependent, with increased directionality with increasing voltage. This may provide new treatment modalities for Acanthamoeba keratitis.
This study shows that an electric field can guide directional migration of Acanthamoeba castellanii which causes Acanthamoeba keratitis. Our finding may provide new treatment modalities for this rare, but potentially devastating disease.
Acanthamoeba; directional cell migration; electric fields