bluuebunny/crossref_metadata_2025 huggingface dataset:
Ocular morbidity Brazilian study: causes of visual impairment
Authors: VERONESE RODRIGUES ML , ARRUDA MELLO PA , COSTA PASSOS AD , REY DE FARIA MA , SOUZA CARVALHO R , KARA JOSÉ N , COLLABORATIVE GROUP N
Published: August 2012

Purpose To determined the causes of visual impairment among patients seeking ophthalmic treatment, in secondary and tertiary level outpatients clinics, in Brazil.

Methods The authors and the Collaborating Groups recorded the main complaints and diagnoses of all the new cases attended at 29 services that maintain Courses of Specialization in Ophthalmology accredited by the Brazilian Council of Ophthalmology (CBO) during a typical week for each one of them.

Results Twenty‐nine centers from ten states in the five regions of the country participated, with a total 3,997 new cases registered and included in the study. Median patient age was 49 years and 42.9% were males. The most frequent complaint (50.1%) was gradual/chronic reduction of visual acuity. The frequency of visual deficiency (low vision and blindness) was 20,3%, and the main causes were Cataract (34.7%), Diseases of the retina (18.0%), Glaucoma (8.0%), Diseases of the Cornea (7.3%), Diseases of the Conjunctiva (5.3%) and non‐corrected Refractive Errors (4.9%). The bilateral blindness rate, among the 3,997 participants, was 3.10% and the main determinants were Cataract(36.3%), Diseases of the Retina (25.0%) and Glaucoma (6.5%).

Conclusion As expected, the frequency of visual impairment in patients seeking eye care was higher than in general population, of regions with similar level of development. The study provided indicators for the establishment of ocular health actions and for the prevention of blindness in Brazil.
Activity and epidemiology of a recently‐opened ophthalmic emergency center in an University Hospital
Authors: AMELOOT F , AGRINIER N , MAALOUF T , ANGIOI K
Published: August 2012

Purpose To investigate characteristics of ophthalmic emergencies in a new unit of emergency care in an University hospital in order to improve the management of patients.

Methods Prospective monocentric cross‐sectional study of ophtalmic emergencies over a 2‐month period. All patients presenting themselves have been included. Patients characteristics, waiting‐time, pathology, and the reality of the emergency were analysed.

Results 1506 patients were examined (average 24 per day). Mean age was 47 years and 60% were male. The median of waiting time was 30 minutes and 24% of patients were registered during the on‐call time. The main reasons for consultation were pain (37%), red eye (32%), vision loss (24%). The main diagnoses were ocular trauma (29%), ocular inflammation and infection (24%), normal examination and refractive error (13%). We have estimated that 38% of our patients did not need an ophthalmic specialized emergency examination or treatment. Numerus and various daily ophtalmic emergencies require relevant diagnosis and treatment. Better information of the population and prevention of trauma and infection could help to decrease the number and the severity of ophtalmic emergencies. An improvement of the general and emergency practitioners knowledges in regards to ophthalmic pathologies could allow them to diagnose and treat begnin cases.

Conclusion There is a real need for a permanent ophthalmic emergency department. However patient education and an improvement of care network could allow us to focus on cases that require specialized cares.
Diabetic retinopathy in Greek Caucasian type 2 diabetic patients, relationship with polymorphism in the plasminogen activator inhibitor 1 and 2 genes
Authors: SOKOLAKIS TH , TSIRONI E , DARDIOTIS E , CHATZOULIS E , ZINZARAS E
Published: August 2012

Purpose Diabetic retinopathy is a sight thratening chronic complication of diabetes and the leading cause of acquired blindness in adults.Population and family studies showed that pathogenesis of diabetic retinopathy depends upon the interaction of several environmental and genetic factors. Plasminogen activator inhibitor 1 and 2 is the major inhibitor of fibrinolysis and gene polymorphism has been related to hypofibrinolysis.

Methods A case control study was carried out.Assesment of diabetic retinopathy was performed by ophthalmoscopy and fluoroangiography when indicated.The comparison of the groups of patients was performed according to the presence or absence of diabetic retinopathy.In each patient the genotype of pai‐1 and pai‐2 was determined using PCR and RFLP techniques.

Results 246 controls & 352 cases observed SERPINB2 Statistical significance of the observed linkage disequilibrium: rs2070682 rs1050813 rs2227690 rs2227692 rs2227667 1.55e‐15 3.55e‐15 1.11e‐16 2.22e‐16 rs2070682 ‐ 2.22e‐16 1.78e‐15 0 rs1050813 ‐ ‐ 2.04e‐12 2.17e‐7 rs2227690 ‐ ‐ ‐ 3.02e‐8 Global result: Fisher's p value is 0.573984 Pearson's p value is 0.573939 SERPINE1 Statistical significance of the observed linkage disequilibrium: rs2070682 rs1050813 rs2227690 rs2227692 rs2227667 1.55e‐15 3.55e‐15 1.11e‐16 2.22e‐16 rs2070682 ‐ 2.22e‐16 1.78e‐15 0 rs1050813 ‐ ‐ 2.04e‐12 2.17e‐7 rs2227690 ‐ ‐ ‐ 3.02e‐8 Global result: Fisher's p value is 0.573984 Pearson's p value is 0.573939

Conclusion Based on the primary analysis derived some important relationship between gene polymorphism and diabetic retinopathy that even last the bonferroni correction.
The Lid Wiper – a specialized structure at the inner eyelid margin for distribution of the tear film
Authors: KNOP E , KORB DR , BLACKIE CA , KNOP N
Published: August 2012

Purpose The lid wiper is an epithelial structure at the inner eyelid border that distributes the thin precorneal tear film during the up‐phase of the blink. Clinically it is the first area of the ocular surface that shows epithelial alterations in dry eye disease.

Methods A literature review on the posterior lid margin and lid wiper is discussed together with own data and considerations of the relevance of the lid wiper region in ocular surface physiology and pathology.

Results The posterior side of the inner lid border has an epithelium of conjunctival morphology with goblet cells. It forms an elevation of about 100µm (10‐12 cell layers) thickness immediately at the crest of the inner border with a slope towards the subtarsal fold. The lid wiper hence forms a distinct epithelial lip that is the only region of the upper lid in contact with the globe as verified by functional tests. It extends along the upper and lower eyelid margins, has a width of typically about 1mm, narrower in the middle and wider to the temporal and nasal sides. The lid wiper of the upper lid appears as a suitable device to distribute the thin precorneal tear film during the blink as supported by the finding that in conditions with increased friction, such as dry eye disease and contact lens wear, the lid wiper is the first zone of the ocular surface that shows a pathological vital staining indicating epithelial destruction.

Conclusion The lid wiper is of underestimated importance for tear film distribution and ocular surface integrity as well as a sensitive early indicator of dry eye disease. Integration and scoring of lid wiper epitheliopathy in dry eye diagnosis and therapy schemes could be a valuable addition to clinical practice. Support DFG KN317/11
Lubricin: translating an idea into a cure
Authors: SULLIVAN DA , SCHMIDT T , KNOP E , KNOP N , SULLIVAN BD
Published: August 2012

Purpose We hypothesize that lubricin, a boundary lubricant, is transcribed, translated and expressed by the ocular surface and serves to protect the cornea and conjunctiva against significant shear forces generated during an eyelid blink. We also hypothesize that lubricin deficiency increases shear stress and promotes corneal damage. Our objective was to test these hypotheses.

Methods Human tissues and cells were processed for molecular, immunochemical and/or biomechanical procedures. Techniques included the use of real‐time PCR, microarrays, sequencing, immunohistochemistry and friction analyses. Methods also involved the corneal evaluation of wildtype and lubricin knockout (KO) mice.

Results Our results show that lubricin is produced by human corneal and conjunctival epithelial cells. Lubricin absence in KO mice is associated with a significant increase in corneal fluorescein staining. Our findings also demonstrate that lubricin functions as a very effective friction‐lowering boundary lubricant at the human cornea‐eyelid interface.

Conclusion Our data indicate that lubricin acts as a natural boundary lubricant to reduce shear stress at the ocular surface. We thank S Richards, S Liu, A Sahin, R Rahimi Darabad and W Kam (Boston) and S Morrison (Calgary) for their help with these studies. This research was supported by NIH grant R01EY05612, the Margaret S. Sinon Scholar in Ocular Surface Research fund, the Canadian Natural Sciences and Engineering Research Council & the Centre for Bioengineering Research and Education at the University of Calgary.

Commercial interest
A new matrix therapy agent in the treatment of corneal ulcers resistant to conventional treatments
Authors: COCHENER B , MURAINE M
Published: August 2012

Purpose To report the cases of a series of patients suffering from corneal ulcers resistant to conventional therapies and treated with a new ophthalmologic solution based on ReGeneraTing Agent technology (RGTA, Cacicol®).

Methods Twenty four patients with corneal ulcer, most often chronic, were included in this opened series. Eleven patients had a neurotrophic ulcer (5 post‐infectious keratitis, 3 chemical burns, 1 Lyell syndrome, 2 others) with corneal anesthesia and persisting despite a 15‐day treatment with only unpreserved artificial tears. Other patients (n=12) had a chronic corneal ulcer of various etiologies (including 2 chemical burns, one associated with neurotrophic keratitis, one Moreen ulcer), and one patient had a severe confluent keratitis. These 13 patients were previously treated with classical lachrymal substitutes, some of them also with topical cyclosporine, corticoids, and/or A vitamin ocular ointment. A total of 11 patients had already received one or several amniotic membrane grafts, without success. All Patients were treated with Cacicol® at a dose regimen of one drop daily every 2 or 3 days for one to 3 months depending on healing.

Results Complete healing was observed for 16 patients, including 7 out of the 11 patients with neurotrophic ulcer, i.e. a cure rate of 67%. However, a large variation has been noted in the time period recovery of corneal surface integrity going from few days to few weeks. An analysis of potential factors that could influence the healing process will be provided. For other patients, 5 failures and 3 improvements without complete healing were reported.

Conclusion Cacicol® appears as a new interesting healing eye drops in the context of severe corneal ulcers resistant to conventional therapies. Its efficacy remains to be proven in randomized double‐blind studies.
Optical coherence tomography: signal signature on neuronal ageing and blood‐retinal barrier status
Authors: BERNARDES R , SANTOS T
Published: August 2012

Purpose We aim to identify the features that allow the discrimination between healthy controls and diabetic patients with optical coherence tomography (OCT) data from the human retina.

Methods Prior studies from our research group showed the association between the status of the blood‐retinal barrier and the consequent changes on the optical characteristics of the retina. Following the same rationale, we have demonstrated the possibility to discriminate healthy controls and diabetic patients by age group and healthy controls from diabetic subjects even though no visible changes could be identified, using a spectral domain OCT, Cirrus HD‐OCT (Carl Zeiss Meditec, Dublin, CA, USA), to gather the refractive index data in between the inner limiting membrane and the retinal pigment epithelium, and the support vector machine (SVM) for the automatic classification. In the work herewith presented, we applied a backward elimination process aiming to reduce the number of features used while simultaneously increase the classification performance.

Results In this work we were able to reduce the number of features used in the SVM algorithm from the initial 46 features down to 10, while simultaneously increase the accuracy up to 97%. Moreover, the different feature set used for the different classifications suggest the presence of specific signatures within OCT data distribution.

Conclusion These findings also suggest the possibility to use this noninvasive imaging modality to assess the neuronal condition and blood‐retinal barrier status.
High‐resolution spectral domain optical coherence tomography findings in reticular pattern dystrohy
Authors: ZERBIB J , MASSAMBA N , QUERQUES G , BLANCO‐GARAVITO R , SROUR M , BONNET C , SOUIED EH
Published: August 2012

Purpose To analyze specific outer retinal and retinal pigment epithelium (RPE) features in reticular pattern dystrophy using spectral‐domain optical coherence tomography (SD‐OCT).

Methods Consecutive patients with reticular pattern dystrophy underwent a complete ophthalmologic examination, including assessment of best‐corrected visual acuity (BCVA), fundus biomicroscopy, and SD‐OCT (Spectralis SD‐OCT, Heidelberg Engineering). Outer retinal and RPE macular features, as evaluated by SD‐OCT (scan passing through the fovea) were analyzed by 3 authors (JZ, NM and GQ).

Results 24 eyes of 13 patients (5 males, 8 women, mean age 71.4) were included. Mean visual acuity was measured at 0.30 (logMAR). The RPE layer in the foveal area appeared normal in 41.7% of scans, while small RPE elevations and RPE bumps were detected in 33.3% and 20.8% of scans, respectively. Inner segment/outer segment (IS/OS) junction appeared disrupted in 50% of scans, and absent in 58.3% of scans. SD‐OCT showed a slight IS/OS elevation in 54.2% of scans. The external limiting membrane (ELM), appeared disrupted in 50% of scans, absent in 37.5% of scans, and elevated in 66.7% of scans. Hypereflective subretinal material accumulation or hyporeflective subretinal lesions, were detected in 33.3% and in 12.5% of scans, respectively. SD‐OCT showed hyporeflective retinal pseudocysts in 16.7% of scans.

Conclusion In this series of reticular pattern dystrophy, SD‐OCT showed disruption of RPE layer and RPE bumps. Outer retinal changes included absence and disruption of both IS/OS junction and ELM. Hypereflective subretinal material accumulation or hyporeflective subretinal lesions, and hyporeflective retinal pseudocysts were also noticed.
Assessment of the spectralis spectral domain oct segmentation software in a retrospective cohort study of exudative amd patients
Authors: TILLEUL J , QUERQUES G , CANOUI‐POITRINE F , LEVEZIEL N , SOUIED EH
Published: August 2012

Purpose To assess the ability of the Spectralis optical coherence tomography (OCT) segmentation software to identify the inner limiting membrane and the Bruch’s membrane in exudative AMD patients.

Methods Thirty‐eight eyes of 38 naive exudative AMD patients were retrospectively included. They all had a complete ophthalmologic examination including Spectralis OCT at baseline, at month‐1 and at month‐2. Reliability of the segmentation software was was defined as good if both inner limiting membrane and Bruch’s membrane were correctly drawn.

Results A total of 38 patients charts were reviewed (114 scans). The inner limiting membrane was correctly drawn in 114/114 SD‐OCT scans (100 %). Conversely, the Bruch’s membrane was correctly drawn in 59/114 scans (51.8 %). The software was less reliable in locating the Bruch’s membrane in case of pigment epithelium detachment (PED) than without PED (42.5% vs 73.5 % respectively, p =0.049).

Conclusion Segmentation of inner limiting membrane was constantly trustworthy but Bruch’s membrane segmentation was poorly reliable using the automatic SD‐OCT Spectralis segmentation software. Based on this software, evaluation of retinal thickness may be incorrect, particularly in case of PED.
Meganuclease targeting HSV‐1 limits viral endothelitis in vivo
Authors: CHAPELLIER B , LABETOULLE M , GAILLEDRAT M , GABISON E
Published: August 2012

Purpose The aim of this study is to assess the antiviral property of a meganuclease targeting HSV‐1 in the prevention of HSV endothelitis in a newly develloped rabbit in vivo model.

Methods NZW received intracamerular injection of recombinant adeno‐associated virus equipped with a constitutive expression cassette containing either the meganuclease gene or a non‐coding sequence. After treatment with intravitreal triamcinolone acetonide, HSV‐1 F(1) virus recombinant for LacZ reporter was injected in the inner chamber of the eye,for an analysis 2 days later. Corneal edema, keratic precipitates and the number of contamination plaques were evaluated.

Results In 4 tests with 24 experimental or control corneas, the meganuclease‐treated groups systematically showed a decrease in their average number of contamination plaques while compared to controls (from 15 to 71%). Presence of protein and inflammatory cells in the aqueous humor or at the endothelium surface, as well as the extension of focal edema, was also limited by the endonuclease.

Conclusion Meganuclease gene therapy targeting HSV‐1 DNA restricts degradation of the endothelium initiated by HSV‐1 injection.
The effects of autologous serum eye drops in the treatment of ocular surface diseases : a retrospective study
Authors: BASLI E , LAROCHE L , LAURENT M , DESPIAU MC , BORDERIE V
Published: August 2012

Purpose To evaluate the efficiency of autologous serum eyedrops in the treatment of severe dry eye (DE) and persistent epithelial defects (PED).

Methods Ninety‐seven patients with PED or DE, unresponsive to conventional treatment, were studied. Functional symptoms and slit‐lamp findings were recorded.

Results The mean LogMAR visual acuity significantly improved from 1.81 (20/1291) +/‐ 9 lines before to 1.69 (20/980) +/‐ 6 lines after treatment (p = 0.01). Visual acuity increased by 2 lines or more in 29 out of 97 eyes (30%) and it decreased by 2 lines or more in 15 eyes (15%). Resolution of 3 subjective symptoms was noticed in 14 out of 97 eyes (14%), resolution of 2 in 22 (23%), resolution of 1 in 29 (30%), and no subjective improvement was found in 32 eyes (33%). Among 75 eyes with PED, ulcer healing was observed in 31 (41%) at 1 month and 61 (81%) at 3 months. In multivariate analysis only presence of limbal deficiency significantly influenced the healing time. The average time from treatment onset to ulcer healing was 213 days in eyes with limbal deficiency and 59 days in eyes with no limbal deficiency (p<0.0001). Among 22 eyes with DE, improvement of subjective symptoms was observed in 8 (36%) at 1 month and 12 (55%) at 3 months. At the end of follow‐up improvement of subjective symptoms was observed in 20 (91%) eyes.

Conclusion Cicatrisation of PED and improvement of DE symptoms were observed in most eyes under autologous serum.
Severe corticoresistant Mooren Ulcers: management with Rituximab and peripheral lamellar graft
Authors: BEN MHAMED M , PEREIRA D , DOAN S , COCHEREAU I , GABISON E
Published: August 2012

Purpose Mooren's ulcer is a rapidly progressive, painful, ulcerative keratitis which affects the peripheral cornea. We report 6 severe cases of Mooren Ulcers (4 patients) with corticoresistance favourably responding to Rituximab infusions.

Methods Retrospective Case series

Results Despite systemic intensive steroid (4/4 patients), and Cyclophosphamide (2/4 patients) therapy, the 6 cases of Mooren’s ulcers progressively spread circumferentially and centrally. Perforation occurred in 4/6 affected eyes, treatment included conjunctival resection and peripheral corneal graft. Rituximab biotherapy was associated with the stabilisation and the healing of the corneal lesions. Systemic steroid therapy was tapered then stopped in all patients within 2‐4 months following rituximab therapy.

Conclusion Rituximab, an anti‐CD20 monoclonal antibody, has been successfully used off‐label for treatment of Severe corticoresistant Mooren’s Ulcer.
16S RNA PCR in the diagnosis of bacterial keratitis and endophthalmitis
Authors: ROBERT PY , SABATIER A , GARNIER F , PLOY MC
Published: August 2012

Purpose Classical bacterial culture often fails to detect the infecting agent in bacterial keratitis and endophthalmitis. Our study was designed to evaluate the contribution of 16S RNA amplification using polymerase chain reaction (PCR) and nested PCR, when compared to culture, in the diagnosis of bacterial keratitis and endophthalmitis.

Methods In this prospective study, we included 63 patients (24 bacterial keratitis and 39 endophthalmitis). For every patient, two samples were taken: one for bacterial culture, the other for PCR. PCR and nested PCR 16S rRNA were performed using universal primers (8/27‐1510 for PCR and 91E‐13BS for nested PCR) and the amplicons were thereafter sequenced.

Results Culture offers a sensibility of 25%; associated to first PCR (8/27‐1510), this rate increase to 31,7%, and with nested PCR (91E‐13BS), we had 34,9% of identifications. We have to note that the rate of positive PCR were much better, but the sequencing rate was 59,5% only. For bacterial keratitis, culture was positive in 25% and PCR in 8%. For aqueous humor samples, culture was positive in 32%, PCR in 25% and nested PCR in 20,6%. Sequencing was possible in 76,5% for humor aqueous samples.. For vitreous samples, the culture alone was positive in 9%, PCR in 36,3%, and nested PCR in 36,3%.

Conclusion 16S rRNA PCR is a rapid alternative to culture, sensitive and reproductible.. PCR associated to culture increases the number of positive samples. PCR was found necessary for the microbiological diagnosis in 10% of all cases (PCR+, cultures‐).
SPARCS: a new method of evaluating contrast sensitivity in patients with glaucoma
Authors: SPAETH G , RICHMAN J
Published: August 2012

Purpose To evaluate a new contrast sensitivity instrument as a diagnostic test for glaucoma.

Methods Glaucoma patients, glaucoma suspects, and controls were prospectively evaluated using the Spaeth‐Richman Contrast Sensitivity (SPARCS) method. Testing was performed monocularly in a standardized testing environment. Contrast thresholds for the central, superonasal, superotemporal, inferonasal, and inferotemporal areas of vision were determined. The contrast sensitivity scores for each area of vision and the cumulative scores were then compared to subjects’ visual field and the amount of optic nerve damage, measured by the Disc Damage Likelihood Scale (DDLS) and the vertical cup to disc ratio. The results were analyzed with Spearman coefficients for continuous variables and the Kruskal‐Wallis test for categorical variables.

Results One hundred and eighteen patients with glaucoma, 18 glaucoma suspects, and 125 controls completed the study. The mean SPARCS scores were 63.0 for patients with glaucoma, 71.9 for glaucoma suspects, and 77.1 for controls (p < 0.01). SPARCS had strong correlations with patients’ visual field (r = 0.76, p < 0.01), DDLS (r = ‐0.73, p < 0.01), and vertical cup to disc (r = ‐0.64, p < 0.01).

Conclusion SPARCS was able to discriminate well between patients with glaucoma, glaucoma suspects, and controls. SPARCS had strong relationships with the degree of visual field and optic nerve damage. SPARCS may become a standard technique to assess visual ability in glaucoma patients.

Commercial interest
Evaluation of the flicker defined form test versus matrix, in normal and glaucoma eyes
Authors: MAY F , GIRAUD JM , FENOLLAND J , EL CHEHAB H , SENDON D , FRANCOZ M , RENARD JP
Published: August 2012

Purpose To evaluate a new perimetric test, the Flicker Defined Form (FDF), and to analyze its capacity to detect visual field defect, comparatively to Matrix, in normal subjects and patients treated for primary angle glaucoma (POAG).

Methods Clinical comparative trial including 65 eyes of 35 subjects who performed: clinical examination, measurement of the retinal nerve fiber layer thickness by scanning laser polarimetry GDx‐Pro, standard automated perimetry (SAP) Humphrey SITA 24.2 test, and Matrix 24.2 test; each subject performed SAP, Matrix and FDF two times, and only the second test was used in the study. Subjects were classified in 2 groups. Control group:35 normal eyes, mean age 40,5 years, without any abnormality in GDx‐Pro or SAP. POAG group: 30 eyes, mean age 69,1 years, treated for POAG, and presenting glaucomatous abnormalities in GDx and SAP: 23 early POAG, 4 moderate and 3 advanced.

Results Control group: 37% (13/35 eyes) present defects revealed by FDF, and 6% (2/35) by Matrix. FDF defects are significantly more frequent than Matrix defects (Pearson Chi 2 test: p < 0,01); in 8 cases of these 13, defects are extended more than 1 quadrant. POAG group: 87% (26/30 eyes) present defects revealed by FDF, and 90% (27/30) by Matrix; there is no significant difference between the 2 tests (Pearson Chi 2 test: p > 0,9). All moderate and advanced POAG eyes present defects in the both tests. Eyes without defect (FDF or Matrix) are 7 early POAG with mild SAP defects.

Conclusion In POAG patients, FDF test and Matrix seem to present high sensibilities: 87% and 90%. Inversely, FDF test specificity is very low, 63%, comparatively to Matrix, 94%, probably in relation with a learning effect.
Delivering it: retinitis pigmentosa
Authors: BLACK GCM
Published: August 2012

Purpose Current technologies for delivering gene testing through conventioinal Sanger sequencing are labour‐intensive and expensive. Over recent years, high‐throughput DNA sequencing techniques (next generation sequencing; NGS) have been successfully implemented in a research context. This presentation discusses the use of using in a clinical service context.

Methods We have applied NGS of 105 genes to patients known to be affected by inherited forms of blindness. This is delivered in the setting of a UK National Health Service accredited diagnostic molecular genetics laboratory. The presentation will discuss the ability of an NGS protocol to identify likely disease‐causing genetic variants.

Results Conventional testing is applicable to the minority of patients with inherited retinal disease and identifies mutations in fewer than one in four of those patients tested. The current NGS assay is directed at all patients with such disorders and identifies disease‐causing mutations in 50‐55% a dramatic increase.

Conclusion An NGS approach delivers a step change in the diagnosis of inherited retnial disease and provides precise diagnostic information and extends the possibility of targeted treatments including gene therapy. Importantly it is likely that this approach will develop rapidly in the near future.
Delivering it: Stargardt disease
Authors: NEMETH AH , DOWNES S
Published: August 2012

Purpose To investigate the use of Sanger and Next Generation Sequencing (NGS) in patients with Stargardt Disease and other ABCA4 retinopathies

Methods Comparison of NGS with traditional Sanger sequencing in patients with possible ABCA4 retinopathies and the development of a clinical diagnostics service for inherited retinal degeneration using NGS.

Results When ABCA4 was tested using Sanger sequencing, the mutation detection rate in patients with a clinical diagnosis of STGD disease was ~80%. In patients with other phenotypes compatible with ABCA4 mutations (eg. end‐stage chorioretinal atrophy, Bulls eye maculopathy or Pattern dystrophy) the detection rate was lower but still significant. Evaluation of NGS using ABCA4 as a test system showed that this technology, particularly the Illumina platform, was highly reliable in detecting the mutations found using Sanger sequencing. When NGS was used in a group of patients with Retinitis Pigmentosa or Cone‐Rod Dystrophy, ~ 6% were found to have ABCA4 mutations, illustrating the heterogeneous phenotype of ABCA4 retinopathies and the difficulties in making a clinical diagnosis.

Conclusion Sequencing is the most efficient method of detecting mutations in STGD and will replace screening technologies. Genetic testing in STGD allows clinicians to improve their clinical pattern recognition, facilitate genetic counselling and identify patients for clinical trials. NGS compared to Sanger sequencing has the added advantage that numerous other genes can be sequenced at the same time and is reliable and cost effective, thereby providing an invaluable diagnostic tool. Genetic testing using NGS for genes involved in retinal degeneration is now available as a service through the Oxford laboratory (www.ouh.nhs.uk/geneticslab).
Using it: NGS in the inherited retinal degeneration clinic
Authors: DOWNES S , NEMETH AH
Published: August 2012

Purpose To describe the clinical application of Next Generation Sequencing (NGS) in a retinal dystrophy clinic

Methods The use of NGS in a clinic setting is discussed using case histories from our research using NGS in IRD to illustrate the potential and complexities of NGS testing for the clinician.

Results Inherited retinal degeneration (IRD) is a common cause of visual impairment (prevalence ~1/3500). There is considerable phenotype and genotype heterogeneity, making a specific diagnosis very difficult without molecular testing. With the introduction of NGS huge amounts of genetic information are generated which highlight the importance of careful clinical phenotyping in the proband and family members with follow up segregation studies and functional analysis in many cases.

Conclusion From our experience in the clinical setting it is evident that NGS has a useful role in diagnosis. In our NGS study our mutation detection rate suggests that many patients are likely to have mutations in novel genes and it is clear that resources are needed to carry out careful family studies and appropriate functional analysis.
Investigation of intra‐cellular metabolism by time‐resolved autofluorescence
Authors: SCHWEITZER D
Published: August 2012

Purpose Changes in metabolism are first signs of pathological processes. The extra‐cellular metabolism can be estimated by parameters of microcirculation. The fluorescence of endogenous fluorophores characterizes the intracellular metabolism. Besides of NADH and FAD which act as electron transporter in the respiratory chain, several other fluorophores like lipofuscin and advanced glycation end products, or collagen and elastin form the autofluorescence of the fundus.

Methods The influence of single fluorophores can be determined combining measurements of fluorescence decay after pulse excitation with spectral measurements. Based on fluorescence and lifetime measurements on isolated fluorophores and separated anatomical structures of porcine eyes, a laser scanner ophthalmoscope was modified for Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO). The eye‐ground of human eyes is excited at 448 nm (70 ps FWHM, 80 MHz) and the fluorescence is detected in 2 spectral channels (490‐560 nm and 560‐700 nm) in TCSPC in 1024 time channels.

Results A certain relation was found between exponents of triple‐exponential fit and anatomical structures. The lifetime tau 1 (about 70 ps) corresponds to the fluorescence in retinal pigment epithelium. The lifetime tau 2 (about 400 ps) originates from layers in the neuronal retina (receptors, bi‐polares, ganglion cells). The lifetime tau 3 (about 3ns) is determined by connective tissue in the nerve fiber layer and to a certain degree by the fluorescence of the crystalline lens, too.

Conclusion The changes of tau 2 in the short wavelength spectral range which were found in the neuronal retina for diabetic patients having no signs of diabetic retinopathy,are interpretable as a loss of free NADH and a shift to protein‐bound NADH.

Commercial interest
Retinal oximetry technology
Authors: HARDARSON S
Published: August 2012

Purpose Disturbances in retinal oxygenation are believed to be involved in several eye diseases, among them retinal vessel occlusions, diabetic retinopathy and possibly glaucoma and age‐related macular degeneration. The technology to reliably and non‐invasively measure retinal oxygenation is emerging after decades of development.

Methods Non‐invasive retinal oximetry utilises the fact that the colour of blood changes as oxygen saturation changes. The most reliable measurements can be obtained from the larger retinal vessels, while measurements of retinal capillaries need further development. Several instruments have been built for non‐invasive oximetry. They measure light absorbance of retinal blood vessels at two or more wavelengths and use the result to calculate retinal vessel oxygen saturation.

Results Several research groups have shown that their instruments are sensitive to changes in retinal vessel oxygen saturation and that the measurements are reproducible. Oximetry has successfully been used to detect differences in retinal oxygenation in many diseases as well as responses to treatment or provocation tests. Accurate calibration is, however, difficult.

Conclusion The current retinal oximetry technology can reliably be used to measure changes in retinal vessel oxygen saturation in the same eye with time. Oximetry can also easily measure differences in means between groups. Further development is needed to increase the accuracy of single measurements.

Commercial interest
New developments in optical coherence tomography
Authors: SCHMETTERER L
Published: August 2012

Purpose Optical coherence tomography (OCT) has become a standard tool in imaging the retina, the optic nerve head and the anterior segment of the eye. In the recent years much effort was directed towards improving the acquisition speed and the resolution of the technique. In addition, functional extensions of the technique were presented.

Methods Ultrahigh‐speed swept sources can be used to improve A‐scan rate in OCT, a technique also called optical frequency domain imaging. Increasing the bandwidth can improve longitudinal resolution in OCT and longer wavelengths in the near infrared improve penetration depth. Adaptive optics OCT improves transversal resolution. Functional extensions include measurement of blood flow and oxygenation.

Results High‐speed imaging allows for three‐dimensional imaging of the posterior and anterior segment of the eye. Using adaptive optics volumetric cellular resolution imaging becomes possible. 1050 nm OCT allows for visualization of the sclera and therefore measurement of choroidal thickness. Functional OCT provides insight into retinal metabolism.

Conclusion Improvements and extensions of OCT have been reported that will find its way into clinical routine. This is expected to result in improved understanding of retinal and optic nerve head disease.
Expression of lysophosphatidic acid, autotaxin and acylglycerol kinase
Authors: ABU EL ASRAR A , MOHAMMAD G , NAWAZ MI , SIDDIQUEI M , KANGAVE D , OPDENAKKER G
Published: August 2012

Purpose To measure the levels of LPA and LPA‐producing enzymes, autotaxin (ATX) and acylglycerol kinase (AGK) in the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and to correlate their levels with clinical disease activity and the level of vascular endothelial growth factor (VEGF). In addition, we examined the expression of ATX, AGK and VEGFR‐2 in the retinas of diabetic rats.

Methods Vitreous samples from 42 PDR and 35 nondiabetic patients were studied by enzyme‐linked immunosorbent assay. Vitreous samples and retinas of rats were examined by Western blot analysis.

Results VEGF, LPA and AGK levels in vitreous samples from PDR patients were significantly higher than those in control patients without diabetes (p<0.001 for all comparisons). ATX levels in PDR with active neovascularization and inactive PDR were significantly lower than those in nondiabetic patients (p=0.045). Mean VEGF and AGK levels in PDR with active neovascularization were significantly higher than those in inactive PDR and nondiabetic patients (p<0.001 for both comparisons). A significant correlation was observed between levels of VEGF and levels of AGK in PDR patients (r=0.954, p<0.001). Western blot analysis revealed a significant increase in the expression of AGK and VEGFR‐2 in vitreous samples and the retinas of diabetic rats compared to nondiabetic controls, whereas ATX was significantly downregulated.

Conclusion ATX‐AGK‐LPA signaling axis might be an important player in the development and progression of diabetic retinopathy.
Retinal pathways involved in the control of eye growth and myopia
Authors: MORGAN IAN
Published: August 2012

Purpose To integrate information from studies on gene expression in animal mpdels and from genetic studies on human myopia to define the pathways which are involved in the development of myopia

Methods A literature search of studies on gene expression in animal models of myopia and human genetic studies was carried out using the Medline database

Results Human genetic studies identify two clusters of mutations which are involved in human high myopia. One involves genes which affect photoreceptor and ON‐bipolar cell function in the outer retina. These mutations are associated with congenital stationary night blindness. A second cluster involves genes involved in extracellular matrix structure and function, which may be important in the sclera, which changes markedly during the development of myopia. ON‐bipolar cells play a major role in the control of dopamine release in the retina. Other evidence supports a role for dopamine in the control of eye growth, which suggests a plausible pathway linking the genes involved in the outer retina to control of eye growth. However, the down‐stream effects of changes in dpoamine release are not well‐characterised studies. However, animal studies have suggested that changes in the expression of the early intermediate gene ZENK and changes in glucagon release within the retina may play a role

Conclusion Huamn genetic studies and studies of changes in gene expression in animal models of myopia provide a consistent but still incomplete picture of the pathways involved in the control of eye growth. Further studies on these pathways are of particular importance given that they implicate retinal dopamine release , which links them to the epidemiological evidence that bright light‐ induced dopamine release can prevent the development of myopia in humans
Retinal ganglion cell differentiation and protection using neuronally differentiated human dental pulp stem cells
Authors: HONG S
Published: August 2012

Purpose To investigate whether the neuronally differentiated human dental pulp stem cells (NDhDPSCs) can differentiate into retinal ganglion cells (RGCs) and whether the NDhDPSCs can protect primary mouse RGCs against oxidative stress injury.

Methods Human dental pulp stem cells were harvested and neuronally differentiated using various conditioned media. By immunohistochemistry, western immunoblots, and real‐time RT‐PCR, their cellular characteristics were evaluated and the best culture condition was determined for differentiation into RGC/glia‐like NDhDPSCs. Regarding glial‐like NDhDPSCs, they were cocultured with primary mouse RGCs under oxidative stress injury.

Results In specialized conditioned media, human dental pulp stem cells were differentiated into RGC/glia‐like NDhDPSCs. They expressed RGC/glia‐specfic markers as well as neuronal stem cell markers. When the glia‐like NDhDPSCs were indirectly cocultured with primary mouse RGCs, they protect the RGCs against oxidative stress injury as determined by TUNEL assay.

Conclusion The human dental pulp stem cells may differentiate into RGC‐like NDhDPSCs in a suitable culture condition. And the glia‐like NDhDPSCs can protect primary mouse RGCs against oxidative stress injury in vitro.
Intermembranaceous spaces of human optic nerve intracanal part
Authors: HUSEVA YA , DENISOV SD
Published: August 2012

Purpose Purpose of research ‐ to reveal laws of structure and distribution of intermembranaceous spaces of optic nerve intracanal part.

Methods 43 bone blocks including optic canal with located in them membranes (dura, arachnoidea, pia), ophthalmic artery of people aged from 26 weeks of prenatal development till 75 years were histologically studied. The structure of optic nerve membranes and intermembranaceous spaces in the cranial, transitive and orbital compartments of optic canal were established. The received data were compared to optic canal MRI results of 27 patients (54 optic canals) aged from 2 months till 75 years on the "VISTA" МР‐tomograph 1 T intensity of magnetic field.

Results Wide, uniform, continuous intermembranaceous spaces are characteristic for optic canal cranial compartment, medially and laterally of optic nerve they are more expressed. In optic canal orbital compartment subdural space is the narrowest, non‐uniform on MRI and bone blocks research. The subarahnoidal space is most expressed in cranial compartment of canal laterally of optic nerve above of it, and on MRI medially of nerve it is absent or narrow, non‐uniform, faltering crack (the similar data is received on histologic research). In transitive optic canal compartment there are separate fragments of subarahnoidal space or its absence in different directions from optic nerve (according to histologic research ‐ above of nerve). In orbital compartment this space in the most cases is closed above and below of optic nerve (by results of research of bone blocks ‐ on all circle of optic nerve).

Conclusion The laws of intermembranaceous space structure and their distributions in optic canal were revealed. MRI was effective method in study of intermembranaceous spaces of optic nerve intracanal part.
3D blood vessels segmentation from optical coherence tomography
Authors: GUIMARAES P , RODRIGUES P , BERNARDES R , SERRANHO P
Published: August 2012

Purpose To compute the 3D retinal vascular network, from standard high‐definition optical coherence tomography (HD‐OCT) data.

Methods Abnormal retinal vascular patterns were shown to be related to retinal and cardiac diseases. Studies have been performed using 2D fundus images. However, the obtained measurements may suffer from missing depth information for improved quantitative accuracy. In this work we resorted to Cirrus HD‐OCT data (Carl Zeiss Meditec, Inc., Dublin, CA, USA) to scan the human macula. Our approach to obtain vessel positioning (depth‐wise) takes advantage of the 2D automatic vessel segmentation of an OCT fundus image. To locate vessels in depth, we compare A‐scans where in the fundus image no vessels were detected to neighbor A‐scans containing vessels. Hence, we are able to identify the location of particular landmarks that characterize the presence of a vessel, such as, the hyper‐reflectivity and the shadowing effect (due to the light absorption by blood).

Results Our algorithm is able to locate both above‐mentioned vessel markers (hyper‐reflective region and shadow). Preliminary data shows promising results. Both markers present good robustness and coherence, as demonstrated by the smoothness of vessels across different B‐scans and the possibility to discriminate between different depth paths of crossing vessels.

Conclusion The findings suggest the possibility to compute the 3D vascular network, noninvasively, using a standard high‐definition OCT. Additionally, accounting for vessel depth will lead to improved measurements of retinal vascular network properties and therefore to possible better correlations between its shape, location, and disease status.
Follow‐up study of MEWDS using adaptive optics retinal imaging
Authors: GOCHO‐NAKASHIMA K , AUDO I , MOHAND‐SAID S , KAMEYA S , YAMAKI K , TAKAHASHI H , SAHEL JA , PAQUES M
Published: August 2012

Purpose To study the effects of pathological damage in cone photoreceptor outer segments (OS) on the visibility of cones in adaptive optics (AO) images.

Methods Four patients with multiple evanescent white dot syndrome (MEWDS) underwent multimodal retinal imaging using optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO). AO images were acquired with a field of 4 x 4 deg at eccentricities ranging from 0 to 8 deg from fovea using an infrared flood‐illumination AO camera (rtx1, Imagine Eyes, France). Cone phoreceptor density was measured and analyzed in comparison to the findings from the other imaging modalities. The procedure was repeated at a follow‐up visit after one month. One patient had 3 additional follow‐up examinations over a 10‐month time.

Results OCT images showed irregularities and disruptions in the OS tip line. Local thinning of the OS layer was observed in several areas of the macula. AO images revealed marked decreases in the density of detectable cones in the exact same regions. The measured cone density was superior to 30000 cells per mm2 in the areas of normal OS length and dropped to less than 10000 cells per mm2 in the regions of shortened OS. The locations of cone mosaic defects in the AO images also corresponded with hypo‐fluorescent areas observed in the late phase of ICG angiography. During clinical recovery, the progressive return of the IS/OS and the OS tip lines to a normal layered structure in OCT images was correlated to an increase in cone density in AO images.

Conclusion AO imaging facilitates detection and monitoring of damage and recovery of the photoreceptor OS during MEWDS. The visibility of cones in AO images seems to be strongly dependent on OS structure and length.
Assessment of different methods for inter‐individual registration of OCT topography for statistical analysis
Authors: HALDAR S , DAVIES N
Published: August 2012

Purpose Assessment of OCT topography is subjective. A statistical method of analysis would be helpful to aid interpretation. This requires the generation of accurate normative topography which in turn requires accurate alignment of normal OCTs. This study assesses 9 methods of alignment.

Methods Normal topography maps were exported from a spectral domain OCT system. Code was written to perform image registration using different methods: User selection of foveal centre, cross‐correlation to a difference of two Gaussian macula template, finding of the thinnest central point and automatic fovea‐finding using Gaussian convolution and centroiding for the red, green and blue channels of the image respectively.

Results Data from 127 left and 110 right eyes were analysed. Mean and total standard deviation across the central 400x400 pixels of the aligned maps were calculated. The lowest standard deviation was achieved by the cross‐correlation method (38.9 microns), followed by the blue channel centroiding method (40.4 microns), the thinnest central point (40.5 microns) and the user‐selected foveal centre (40.6 microns). Convolution with a Gaussian to identify the fovea produced the worst results with mean SD of 49.9‐64.8 microns.

Conclusion Cross‐correlation with a difference of two Gaussian macula template appears superior for inter‐individual topography registration in OCT in comparison with fovea‐finding methods. Blue centroid and thinnest point were the best other methods. The cross‐correlation technique will lead to the most accurate normal maps for statistical comparison with data from pathological OCT topography.