The entry point of the central retinal artery into the outer meningeal sheath of the optic nerve.

INTRODUCTION: The entry point of the central retinal artery (CRA) into the outer meningeal sheath of the optic nerve posterior to the globe has been studied and debated for more than one hundred years. The authors have supervised an orbital anatomy course for more than two decades. This article summarizes previous studies of the CRA and presents the results of dissections of 67 orbits. MATERIALS AND METHODS: Heads were hemisected prior to dissection at the Vagelos College of Physicians and Surgeons of Columbia University. The authors measured the entry point of the CRA with a caliper and noted the meridional orientation of the CRA. RESULTS: The mean entry point was 10.65 mm posterior to the globe, with a range of 5 to 18 mm. Most commonly, the CRA entered the sheath in the inferior meridian, but some entered slightly inferomedially or inferolaterally. CONCLUSIONS: The entry point of the CRA into the sheath of the optic nerve is variable, and without detailed angiography the clinician cannot know the course of the CRA prior to performing invasive intraorbital procedures. Knowledge of common variations in CRA entry into the outer meningeal sheath of the optic nerve should help to minimize injury during surgery.

Micro-staining microbes: An alternative to traditional staining of microbiological specimens using microliter volumes of reagents.

Microbial staining techniques are widely employed in clinical and academic laboratories for classifying and identifying microorganisms derived from clinical, food and environmental samples. Staining allows for the rapid visualization and determination of many morphological characteristics of microorganisms, used for their identification and classification. Over the past century, staining techniques such as the Gram stain, the Capsule stain, the Acid-fast stain and the Endospore stain, have seen few advances, and manual staining remains the gold standard. Typical instructions for these staining procedures recommend 'flooding' glass slides with milliliter volumes of dye, resulting in large volumes of hazardous waste. Here we present micro-staining, a simple alternative to flooding that utilizes microliter volumes of dye. Micro-staining minimizes the volume of waste generated, leads to significant cost savings for the laboratory, requires limited training, and produces results with equivalent quality to traditional stains.

Intraretinal Correlates of Reticular Pseudodrusen Revealed by Autofluorescence and En Face OCT.

Purpose: We sought to determine whether information revealed from the reflectance, autofluorescence, and absorption properties of RPE cells situated posterior to reticular pseudodrusen (RPD) could provide insight into the origins and structure of RPD. Methods: RPD were studied qualitatively by near-infrared fundus autofluorescence (NIR-AF), short-wavelength fundus autofluorescence (SW-AF), and infrared reflectance (IR-R) images, and the presentation was compared to horizontal and en face spectral domain optical coherence tomographic (SD-OCT) images. Images were acquired from 23 patients (39 eyes) diagnosed with RPD (mean age 80.7 ± 7.1 [SD]; 16 female; 4 Hispanics, 19 non-Hispanic whites). Results: In SW-AF, NIR-AF, and IR-R images, fundus RPD were recognized as interlacing networks of small scale variations in IR-R and fluorescence (SW-AF, NIR-AF) intensities. Darkened foci of RPD colocalized in SW-AF and NIR-AF images, and in SD-OCT images corresponded to disturbances of the interdigitation (IZ) and ellipsoid (EZ) zones and to more pronounced hyperreflective lesions traversing photoreceptor-attributable bands in SD-OCT images. Qualitative assessment of the outer nuclear layer (ONL) revealed thinning as RPD extended radially from the outer to inner retina. In en face OCT, hyperreflective areas in the EZ band correlated topographically with hyporeflective foci at the level of the RPE. Conclusions: The hyperreflective lesions corresponding to RPD in SD-OCT scans are likely indicative of degenerating photoreceptor cells. The darkened foci at positions of RPD in NIR-AF and en face OCT images indicate changes in the RPE monolayer with the reduced NIR-AF and en face OCT signal suggesting a reduction in melanin that could be accounted for by RPE thinning.

Intracellular Position of Centrioles and the Direction of Homeostatic Epithelial Cell Movements in the Mouse Cornea.

Corneal epithelial cells exhibit continuous centripetal movements at a rate of about 30 µm per day, but neither the driving force nor the mechanism that determines the direction of movements is known. To facilitate the investigation of homeostatic cell movement, we examined if the intracellular position of a centriole can be used as a directional marker of epithelial cell movements in the mouse cornea. A direction of cell movements was estimated in fixed specimens from a pattern of underlying subepithelial nerve fibers. Intracellular position of centrioles was determined by gamma-tubulin immunohistology and plotted in a narrow strip along the entire diameter of a cornea from limbus to limbus. When we determined the position of centrioles in the peripheral cornea where cell movements proceed generally along a radial path, about 55% of basal epithelial cells contained a centriole in the front half of a cell. However, in the central cornea where cells exhibit a spiral pattern of movements, centrioles were distributed randomly. These results suggest that centrioles tend to be positioned toward the direction of movement in corneal basal epithelial cells when they are moving centripetally at a steady rate.

A Candidate Gene Association Study Identifies DAPL1 as a Female-Specific Susceptibility Locus for Age-Related Macular Degeneration (AMD).

Age-related macular degeneration (AMD) is the leading cause of blindness among white caucasians over the age of 50 years with a prevalence rate expected to increase markedly with an anticipated increase in the life span of the world population. To further expand our knowledge of the genetic architecture of the disease, we pursued a candidate gene approach assessing 25 genes and a total of 109 variants. Of these, synonymous single nucleotide polymorphism (SNP) rs17810398 located in death-associated protein-like 1 (DAPL1) was found to be associated with AMD in a joint analysis of 3,229 cases and 2,835 controls from five studies [combined PADJ = 1.15 × 10(-6), OR 1.332 (1.187-1.496)]. This association was characterized by a highly significant sex difference (Pdiff = 0.0032) in that it was clearly confined to females with genome-wide significance [PADJ = 2.62 × 10(-8), OR 1.541 (1.324-1.796); males: PADJ = 0.382, OR 1.084 (0.905-1.298)]. By targeted resequencing of risk and non-risk associated haplotypes in the DAPL1 locus, we identified additional potentially functional risk variants, namely a common 897-bp deletion and a SNP predicted to affect a putative binding site of an exonic splicing enhancer. We show that the risk haplotype correlates with a reduced retinal transcript level of two, less frequent, non-canonical DAPL1 isoforms. DAPL1 plays a role in epithelial differentiation and may be involved in apoptotic processes thereby suggesting a possible novel pathway in AMD pathogenesis.

Rare and common variants in extracellular matrix gene Fibrillin 2 (FBN2) are associated with macular degeneration.

Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruch's membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10 337 cases and 11 174 controls (OR = 1.10; P-value = 3.79 × 10(-5)). Thus, it appears that rare and common variants in a single gene--FBN2--can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruch's membrane in maintaining blood-retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes.

Cadherin 5 is regulated by corticosteroids and associated with central serous chorioretinopathy.

Central serous chorioretinopathy (CSC) is characterized by leakage of fluid from the choroid into the subretinal space and, consequently, loss of central vision. The disease is triggered by endogenous and exogenous corticosteroid imbalance and psychosocial stress and is much more prevalent in men. We studied the association of genetic variation in 44 genes from stress response and corticosteroid metabolism pathways with the CSC phenotype in two independent cohorts of 400 CSC cases and 1,400 matched controls. The expression of cadherin 5 (CDH5), the major cell-cell adhesion molecule in vascular endothelium, was downregulated by corticosteroids which may increase permeability of choroidal vasculature, leading to fluid leakage under the retina. We found a significant association of four common CDH5 SNPs with CSC in male patients in both cohorts. Two common intronic variants, rs7499886:A>G and rs1073584:C>T, exhibit strongly significant associations with CSC; P = 0.00012; odds ratio (OR) = 1.5; 95%CI [1.2;1.8], and P = 0.0014; OR = 0.70; 95%CI [0.57;0.87], respectively. A common haplotype was present in 25.4% male CSC cases and in 35.8% controls (P = 0.0002; OR = 0.61, 95% CI [0.47-0.79]). We propose that genetically predetermined variation in CDH5, when combined with triggering events such as corticosteroid treatment or severe hormonal imbalance, underlie a substantial proportion of CSC in the male population.

Reticular pseudodrusen in early age-related macular degeneration are associated with choroidal thinning.

PURPOSE: To compare choroidal thickness (CT) measurements in early AMD between patients with and without reticular pseudodrusen (RPD) using spectral-domain optical coherence tomography (SD-OCT). METHODS: This cross-sectional study examined 84 age- and sex-matched AMD patients (40 RPD [63 eyes], 44 non-RPD [75 eyes]). Fundus photographs and scanning laser ophthalmoscopy images were graded to identify RPD and non-RPD groups by three retinal specialists (MO, SY, SB) who were masked to corresponding SD-OCTs. CT at the fovea and 2400 to 3000 µm superior and inferior to the fovea was measured on SD-OCT by a grader (AG) and reviewed by a retinal specialist (SB). Only images with a clear posterior choroidal margin were analyzed (six eyes excluded due to poor image quality), and enhanced depth imaging SD-OCT was used when available (20 of 138 eyes). Greatest retinal thickness (RT) on horizontal foveal SD-OCT was also recorded. RESULTS: Mean CTs in the superior, foveal, and inferior macula in RPD (191.3 µm ± 57.9 SD, 176.3 µm ± 60.5 SD, 179.7 µm ± 56.24 SD) were significantly less than that of non-RPD (228.0 µm ± 66.1 SD, 216.5 µm ± 70.3 SD, 224.4 µm ± 71.9 SD; P = 0.0010, P = 0.0005, P = 0.0001, respectively), as was greatest RT (P = 0.0301). CONCLUSIONS: CT was thinner throughout the macula in the RPD group as compared with the non-RPD group. The current analysis supports an association between RPD and a thinned choroidal layer and is consistent with a choroidal etiology of RPD. CT may be integral to understanding RPD, and may be helpful in stratifying AMD progression risk.

Occult temporal arteritis in a 54-year-old man.

A 54-year-old white man with a remote history of pars planitis reported transient monocular visual loss (TMVL) in the left eye on standing. The following week he experienced multiple similar episodes. He denied associated systemic symptoms. Initial examination showed old peripheral retinal vascular sheathing and delayed retinal arterial filling time. Complete blood count, erythrocyte sedimentation rate, and MRI studies of the head and neck were normal. One week later, there were multiple cotton wool spots in the posterior pole, a relative afferent pupillary defect, and subtle visual field loss in the left eye. Evaluation for infectious, inflammatory, or embolic etiologies was nonrevealing. Biopsy of the prominent but nontender temporal arteries showed granulomatous inflammation, fragmentation, and duplication of the internal elastic lamina consistent with the temporal arteritis (TA). Radiography and MRI of the chest revealed dilation of the ascending aorta. The patient began treatment with high-dose oral steroids with resolution of his TMVL and retinal cotton wool spots and decrease in the size of the temporal arteries. Our case demonstrates the importance of considering TA in the setting of TMVL, visual loss, cotton wool spots, or dilated nontender temporal arteries in an otherwise asymptomatic patient even with normal inflammatory markers. Long-term follow-up is essential in unusual cases such as this one, given the high risk of ocular and systemic morbidity with TA.

Transmission of light to the young primate retina: possible implications for the formation of lipofuscin.

The purpose of this study was to determine the transmission properties of the anterior segment of young primate eyes and potentially relate those changes to photochemical processes in the retina that lead to the early, rapid formation of lipofuscin. A simple method has been developed to determine the optical properties of the anterior segment of the intact eye. Using this technique, the transmission/absorption properties of primate cadaver eyes were determined. A young primate anterior segment has a maximum absorption at 365nm due to the presence of the O-ß-glucoside of 3-hydroxykynurenine in the lens. This is synthesized in the last trimester of gestation. Although this compound filters out most of the UV light from reaching the retina, there is a small window of transmission centered on an absorption minimum at 320nm. This closes by the second decade of life. The window of transmission of UV light to the primate retina may explain the initial accelerated formation of lipofuscin in the young human retina by a photochemical process. This would be exacerbated by any decrease in the ozone layer with concomitant increase in UV-B reaching the earth's surface.

The behaviour of Drosophila adult hindgut stem cells is controlled by Wnt and Hh signalling.

The intestinal tract maintains proper function by replacing aged cells with freshly produced cells that arise from a population of self-renewing intestinal stem cells (ISCs). In the mammalian intestine, ISC self renewal, amplification and differentiation take place along the crypt-villus axis, and are controlled by the Wnt and hedgehog (Hh) signalling pathways. However, little is known about the mechanisms that specify ISCs within the developing intestinal epithelium, or about the signalling centres that help maintain them in their self-renewing stem cell state. Here we show that in adult Drosophila melanogaster, ISCs of the posterior intestine (hindgut) are confined to an anterior narrow segment, which we name the hindgut proliferation zone (HPZ). Within the HPZ, self renewal of ISCs, as well as subsequent proliferation and differentiation of ISC descendants, are controlled by locally emanating Wingless (Wg, a Drosophila Wnt homologue) and Hh signals. The anteriorly restricted expression of Wg in the HPZ acts as a niche signal that maintains cells in a slow-cycling, self-renewing mode. As cells divide and move posteriorly away from the Wg source, they enter a phase of rapid proliferation. During this phase, Hh signal is required for exiting the cell cycle and the onset of differentiation. The HPZ, with its characteristic proliferation dynamics and signalling properties, is set up during the embryonic phase and becomes active in the larva, where it generates all adult hindgut cells including ISCs. The mechanism and genetic control of cell renewal in the Drosophila HPZ exhibits a large degree of similarity with what is seen in the mammalian intestine. Our analysis of the Drosophila HPZ provides an insight into the specification and control of stem cells, highlighting the way in which the spatial pattern of signals that promote self renewal, growth and differentiation is set up within a genetically tractable model system.

A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila.

The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E) is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E--signaling through a nuclear receptor heterodimer consisting of EcR and USP--induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC) through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.

Oxindolealanine in age-related human cataracts.

The present study was performed in order to obtain structural and quantitative information regarding the modifications that take place in the human lens as a result of tryptophan oxidation. In particular, the early tryptophan oxidation product, oxindolealanine (OIA) has been detected in lyophilized and hydrolyzed cataractous lenses by mass spectrometry. OIA was confirmed in human cataract samples by observing its ion (m/z 221), fragmentation pattern and absorption spectrum. Quantitative results indicate that there are differences in the amounts of OIA in the nucleus versus the cortex in human cataractous lenses. Expressed as a ratio to the level of phenylalanine (Phe), the nucleus has more than one and a half times greater levels of OIA as compared to the cortex [nucleus=(3.7+/-0.7)x10(-2) versus cortex=(2.3+/-0.3)x10(-2)]. Furthermore, the average value for the OIA/Phe ratio in the calf lens (controls) was (0.8+/-0.2)x10(-2) as compared to (3.7+/-0.7)x10(-2) in human cataractous lens nucleus (p<0.05). The quantitative results correspond to a 4.6-fold increase of OIA in human cataractous lenses. In a separate series of experiments using HPLC with photodiode array (PDA) detection only, the differences in OIA levels in cataract nucleus versus cortex and cataracts versus controls closely matched the LC/MS data. The results suggest that OIA levels are elevated in human cataractous lenses thus providing further evidence to implicate tryptophan oxidation in this process.

A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.

Age-related macular degeneration (AMD) is the most frequent cause of irreversible blindness in the elderly in developed countries. Our previous studies implicated activation of complement in the formation of drusen, the hallmark lesion of AMD. Here, we show that factor H (HF1), the major inhibitor of the alternative complement pathway, accumulates within drusen and is synthesized by the retinal pigmented epithelium. Because previous linkage analyses identified chromosome 1q25-32, which harbors the factor H gene (HF1/CFH), as an AMD susceptibility locus, we analyzed HF1 for genetic variation in two independent cohorts comprised of approximately 900 AMD cases and 400 matched controls. We found association of eight common HF1 SNPs with AMD; two common missense variants exhibit highly significant associations (I62V, chi2 = 26.1 and P = 3.2 x 10(-7) and Y402H, chi2 = 54.4 and P = 1.6 x 10(-13)). Haplotype analysis reveals that multiple HF1 variants confer elevated or reduced risk of AMD. One common at-risk haplotype is present at a frequency of 50% in AMD cases and 29% in controls [odds ratio (OR) = 2.46, 95% confidence interval (1.95-3.11)]. Homozygotes for this haplotype account for 24% of cases and 8% of controls [OR = 3.51, 95% confidence interval (2.13-5.78)]. Several protective haplotypes are also identified (OR = 0.44-0.55), further implicating HF1 function in the pathogenetic mechanisms underlying AMD. We propose that genetic variation in a regulator of the alternative complement pathway, when combined with a triggering event, such as infection, underlie a major proportion of AMD in the human population.

Finite element model of cornea deformation.

Cornea surgeons have observed that changes in cornea curvature can follow cataract surgery and cause astigmatism. The placement of surgical incisions has been shown to influence these curvature changes. Though empirical data has been collected about this phenomenon, a biomechanical model has not been employed in predicting post-surgical outcomes. This work implemented an incised finite element model of the eye to investigate factors influencing corneal shape after surgery. In particular, the effects of eye muscle forces and intra-ocular pressure were simulated. Cornea shape change was computed via finite element analysis, and the resulting change in cornea curvature was measured by fitting quadratic curves to the horizontal and vertical meridians of the cornea. Results suggest that these two sources of deforming force counteract each other and contribute to astigmatism in perpendicular directions.

Transmission of light to the aging human retina: possible implications for age related macular degeneration.

The purpose of this study is to determine the transmission properties of the anterior segment of the human eye as a function of age and relate those changes to possible consequences for retinal disorders. For this a new method has been developed. This consists of a probe which is inserted into the posterior sclera and detects light passing through the anterior segment. The probe is connected to a CCD spectrophotometer via a fibre optic bundle. Using this, the transmission properties of human cadaver eyes were determined. A young primate anterior segment has a maximum absorption of 365 nm due to the O-beta-glucoside of 3-hydroxykynurenine (3-HKG) in the lens. There is a steep increase in transmission of the human anterior segment at wavelengths longer than 400 nm. With aging there is an increase in absorption throughout the visible such that by the sixth decade only 20% of blue light is transmitted to the retina compared to the young primate eye. The rate of decrease of blue light was similar to the age related change of the ratio of absorbance at 365/320 nm of the lens. (IOVS 41:1454;1999). The age related rate of decrease in the transmission of blue light to the retina was similar to the rate of increase of lipofuscin formation in the retina, and the amount of light absorbed by A2E in the RPE is constant from the second to seventh decade. Although this yellowing is thought to be detrimental to the lens, it would appear to be beneficial to the retina. It was determined that the implantation of a standard IOL after cataract surgery increased the amount of light absorbed by A2-E by approximately a factor of five.

The effect of incisions for cataract on corneal curvature.

PURPOSE: To determine the magnitude and duration of change on the horizontal and vertical meridians of the cornea after five different incisions for cataract. DESIGN: Retrospective comparative interventional study of five commonly used incisions for cataract surgery: extracapsular cataract extraction (ECCE), 6-mm superior scleral tunnel (6Sup), 3-mm superior scleral tunnel (3Sup), 3-mm temporal scleral tunnel (3Temp), and 3-mm temporal corneal incision (3Cor). PARTICIPANTS: A total of 662 cases with preoperative regular astigmatism, measured with keratometry. METHODS: The mean net change on each meridian was computed at 1 day, 1 week, 2 weeks, 1 month, 1.5 months, 2 months, 4 months, 6 months, and 12 months and at succeeding 6-month intervals after surgery. Best-fit parameters were calculated for the observed changes in the horizontal and vertical keratometry values after each incision. To determine when the cornea stabilized, average change on the horizontal and vertical meridians was compared with an estimate of the accuracy of keratometry measurement. MAIN OUTCOME MEASURES: The pattern of change on the horizontal and vertical meridians and time for the cornea to stabilize after each incision. RESULTS: The initial and final net changes after a superior incision decrease with length. A sigmoid equation describes the course of the changes on the horizontal and vertical meridians after the superior incisions. The changes after the temporal incisions depend linearly on time after surgery. Considering the uncertainty of keratometry, the corneal meridians stabilized 4.5 months after ECCE, 1.2 months after 6Sup, and 0.3 months after 3Sup. No significant change was detected on the horizontal and vertical meridians after 3Temp and 3Cor. CONCLUSIONS: The magnitude and the duration of keratometric change on the horizontal and vertical meridians of the cornea depend on the length and location of the incision. Within the limits of measurement error, no significant change in corneal curvature was detected after either small temporal incision.

Maximum acceptable dose of ultraviolet radiation: a safety limit for cataract.

PURPOSE: To develop a method for experimental estimation of toxicity for continuous dose-response relationships. To apply this method to cataract induced by ultraviolet radiation (UVR) in young rats. METHODS: After establishing experimentally the frequency distribution of light scattering of normal physiologically clear lenses, the lower limit of pathological light scattering is defined such that a certain fraction, for example 97.5%, of normal lenses scatter less light. RESULTS: The dose-response function for UVR and cataract is determined experimentally. With this function, the dose corresponding to the lower limit of pathological light scattering may be determined as the maximum acceptable dose (MAD). The MAD0.975 for UVR 300 nm was determined to be 2.2 kJ/m2. CONCLUSIONS: The method can serve as a basis for establishing safety standards for UVR-induced cataract and probably other continuous dose-response functions.