Diabetic macular edema : cases and diagnostics

In the new book series 'ESASO Course Series', the essentials of the courses of the European School for Advanced Studies in Ophthalmology (ESASO) are made available to interested ophthalmologists, optometrists, technicians and residents all over the world. In this volume, the seminars on surgical retina presented by renowned experts during ESASO's activities are collected. Many specialists have contributed their knowledge to make this volume a device to give practical support. The topics range from prevention to state-of-the-art diagnostic techniques and the latest surgical treatment options for many eye conditions such as diabetic retinopathy and rhegmatogenous retinal detachment. This publication provides the ophthalmologist with the main aspects of surgical retina in a simple and practical update.

Bax-induced apoptosis in Leber's congenital amaurosis: a dual role in rod and cone degeneration.

Pathogenesis in the Rpe65(-/-) mouse model of Leber's congenital amaurosis (LCA) is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65(-/-) mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65), was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65(-/-) mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors.Quantitative PCR analysis showed that increased expression of pro-apoptotic Bax and decreased level of anti-apoptotic Bcl-2 were restored in Rpe65(-/-)/Gnat1(-/-) mice lacking the Gnat1 gene encoding rod transducin. Moreover, photoreceptor apoptosis was prevented as assessed by TUNEL assay. These data indicate that abnormal activity of opsin apoprotein induces retinal cell apoptosis through the Bcl-2-mediated pathway. Following immunohistological and real-time PCR analyses, we further observed that decreased expression of rod genes in Rpe65-deficient mice was rescued in Rpe65(-/-)/Bax(-/-) mice. Histological and TUNEL studies confirmed that rod cell demise and apoptosis in diseased Rpe65(-/-) mice were dependent on Bax-induced pathway. Surprisingly, early loss of cones was not prevented in Rpe65(-/-)/Bax(-/-) mice, indicating that pro-apoptotic Bax was not involved in the pathogenesis of cone cell death in Rpe65-deficient mice.This is the first report, to our knowledge, that a single genetic mutation can trigger two independent apoptotic pathways in rod and cone photoreceptors in Rpe65-dependent LCA disease. These results highlight the necessity to investigate and understand the specific death signaling pathways committed in rods and cones to develop effective therapeutic approaches to treat RP diseases.

Mutation in the human homeobox gene NKX5-3 causes an oculo-auricular syndrome.

Several dysmorphic syndromes affect the development of both the eye and the ear, but only a few are restricted to the eye and the external ear. We describe a developmental defect affecting the eye and the external ear in three members of a consanguineous family. This syndrome is characterized by ophthalmic anomalies (microcornea, microphthalmia, anterior-segment dysgenesis, cataract, coloboma of various parts of the eye, abnormalities of the retinal pigment epithelium, and rod-cone dystrophy) and a particular cleft ear lobule. Linkage analysis and mutation screening revealed in the first exon of the NKX5-3 gene a homozygous 26 nucleotide deletion, generating a truncating protein that lacked the complete homeodomain. Morpholino knockdown expression of the zebrafish nkx5-3 induced microphthalmia and disorganization of the developing retina, thus confirming that this gene represents an additional member implicated in axial patterning of the retina.

Identification of HMX1 target genes: a predictive promoter model approach.

PURPOSE: A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway's function, we sought to identify the target genes. METHODS: We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. RESULTS: The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. CONCLUSIONS: Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1.

Remaining rod activity mediates visual behavior in adult Rpe65-/- mice.

Purpose: C57/Bl6, Cpfl1-/- (Cone photoreceptors function loss 1; pure rod function), Gnat1alpha-/- (rod alpha-transducin; pure cone function) and Rpe65-/-;Rho-/- double knock-out mice were studied in order to distinguish the respective contributions of the different photoreceptor (PR) systems that enable light perception and mediate a visual reflex in adult Rpe65-/- mice using a simple behavioural procedure. Methods: Visual function was estimated using a rotating automatized optomotor drum covered with vertical black and white stripes at spatial frequencies of 0.025 to 0.5 cycles per degree (cpd) in both photopic and scotopic conditions. To evaluate the contribution as well as the light intensity threshold of each PR system, we tested the mouse strains with different luminances. Results: Stripe rotation elicits head movements in wild-type (WT) animals in photopic and scotopic conditions depending on the spatial frequency, whereas Cpfl1-/- mice show a reduced activity in the photopic condition and Gnat1alpha-/- mice an almost absent response in the scotopic condition. Interestingly, a robust visual response is obtained with Rpe65-/- knockout mice at 0.075 cpd and 0.1 cpd in the photopic condition. The residual rod function in the Rpe65-/- animals was demonstrated by testing Rpe65-/-;Rho-/- mice that present no response in photopic conditions. Conclusions: The optomotor test is a simple method to estimate the visual function, and to evaluate the respective contributions of rod and cone systems. Using this test, we demonstrate that in Rpe65-/- mice, devoid of functional cones and of detectable 11-cis-retinal protein, rods mimic in part the cone function by mediating vision in photopic conditions.

CCR2/CCL2-mediated inflammation protects photoreceptor cells from amyloid-β-induced apoptosis.

Age-related macular degeneration is characterized by the formation of drusen containing amyloid-β (Aβ) and the degeneration of photoreceptors. To explore the largely unknown role of Aβ in the retina, we investigated the effects on photoreceptors of the oligomeric form of Aβ(1-42). Subretinal injection of the Aβ peptide induced misplaced expression of recoverin and synaptophysin in the photoreceptors, oxidative stress in their inner and outer segments, and finally apoptosis. Aβ did not induce cell death in purified photoreceptor cell cultures, but did so in retinal cell cultures, thereby suggesting that the cellular environment plays a role in Aβ-induced photoreceptor apoptosis. Subretinal injection of Aβ was followed by activation and migration of microglial cells and then by photoreceptor apoptosis. Microglial cells phagocytosed rhodopsin-containing debris and Aβ in the subretinal space. Quantitative RT-PCR allowed us to identify a specific gene expression profile associated with the Aβ-induced progression of retinal degeneration and consistent with oxidative stress, inflammation, and an apoptotic program. The gene most highly upregulated in Aβ-injected retinas was that for the chemokine CCL2, and its absence or that of its cognate receptor CCR2 greatly reduced migration of activated microglial cells to the site of retinal injury and profoundly worsened photoreceptor degeneration and disorganization of the retinal pigment epithelium in Aβ-injected retinas. Our study pinpoints the roles of Aβ and of CCL2/CCR2 axis-dependent inflammation in photoreceptor apoptosis.

Photodynamic therapy for polypoidal choroidal vasculopathy.

The first effective therapy for exudative macular degeneration (AMD) was Photodynamic Therapy (PDT). Diagnosis of the disease was to a large extent by fluorescein angiography (FA). Distinguishing between the leaky choroidal neovessels (CNV) associated with exudative AMD, and the polypoidal structures associated with Polypoidal Choroidal Vasculopathy (PCV) is not always easy using FA alone. The switch to Indocyanine Green angiography helped to pinpoint PCV, and thus to study the efficacy of photodynamic therapy of this particular form of retinal disease, which is more frequently encountered among pigmented individuals. The results appear to be quite promising, and in the year following treatment only a small fraction of the patients had to be retreated. Alternatively, treating PCV with repeated intravitreal VEGF blocking agents was not as successful as it was in the treatment of wet AMD. However, combining PDT-induced angio-occlusion of the polypoidal lesions with anti-vascular endothelial growth factor therapy was shown to be quite effective, and the combination of PDT with an anti-angiogenic agent as well as a steroid, in a triple therapy, was recently also shown to be a quite promising option. In the present article we review the data on PDT of PCV, including combination therapies and alternative treatments. We also report on similarities and differences between AMD and PCV.

Further delineation of the facial 13q14 deletion syndrome in 13 retinoblastoma patients.

Thirteen years ago, Motegi and colleagues (J Med Genet 1987;24:696-697) summarized the specific facial phenotype of six Japanese retinoblastoma patients with interstitial 13q14 deletions. Among a series of 228 propositi with retinoblastoma referred to the Lausanne Retinoblastoma Clinic for treatment and genetic counseling between 1986 and 1997, 13 (5.7%) were diagnosed with a cytogenetic de-novo 13q14 deletion. We confirm the presence of the reported facial phenotype in our population of Caucasian patients and describe additional clinical traits, thus extending the facial phenotype associated with the 13q14 deletion. Del(13q14) comprises, among others, cranial anomalies, frontal bossing, deeply grooved and long philtrum, depressed and broad nasal bridge, bulbous tip of the nose, thick lower lip, thin upper lip, broad cheeks, and large ears and lobules. Recognition of this particular facial appearance was instrumental in the genetic diagnosis of 13q deletions and in the presymptomatic diagnosis of retinoblastoma in a significant number of our cases. Identification of this phenotype in a retinoblastoma patient allows for efficient diagnosis of recurrence in his progeny and/or sibship, while its ignorance will compromise genetic counseling due to the possible difficulties in detecting large deletions by standard molecular mutation analysis. Recognition of this syndrome in newborns without known familial risk for retinoblastoma is even more important as it is a clear warning sign that indicates immediate ophthalmic examination.

Identification of the minimal promoter region of the mouse NKX5-3, a transcription factor implicated in eye development.

Early ocular development is controlled by a complex network of transcription factors, cell cycle regulators, and diffusible signalling molecules. Together, these molecules regulate cell proliferation and apoptosis, and specify retinal fate. NKX5-3 is a homeobox transcription factor implicated in eye development. The analysis of the 5'-flanking region of the mouse Nkx5-3 gene revealed a predicted TATA-less promoter sequence between -416 and -166 of the translation start site. To functionally characterise Nkx5-3 promoter activity, serial deletions of the promoter sequence were introduced in pGL-3 basic vector and promoter activity of these 5'- and 3'-deleted constructions was tested in HeLa and CHO cells. Transactivation assays identified a region between -350 and -296 exhibiting promoter-like activity. Combined analysis by deletions and point mutations showed that this sequence, containing multiple Sp1 binding sites was necessary to promote transcriptional activity. Binding of Sp1 to this region was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation, using an antibody specific for Sp1. Altogether, these results demonstrated that the immediate upstream region of Nkx5-3 gene possessed a strong intrinsic promoter activity in vitro, suggesting a potential role in Nkx5-3 transcription in vivo.

Macular dystrophy associated with the mitochondrial DNA A3243G mutation: pericentral pigment deposits or atrophy? Report of two cases and review of the literature.

BACKGROUND: The A3243G point mutation in mitochondrial DNA (mtDNA) is associated with MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and MIDD syndromes (maternally inherited diabetes and deafness). Both MELAS and MIDD patients can present with visual symptoms due to a retinopathy, sometimes before the genetic diagnosis is made. CASE PRESENTATION: Patient 1: 46 year-old woman with diabetes mellitus and hearing loss was referred for an unspecified maculopathy detected during screening evaluation for diabetic retinopathy. Visual acuity was 20/20 in both eyes. Fundus examination showed bilateral macular and peripapillary hyperpigmented/depigmented areas.Patient 2: 45 year-old woman was referred for recent vision loss in her left eye. History was remarkable for chronic fatigue, migraine and diffuse muscular pain. Visual acuity was 20/20 in her right eye and 20/30 in her left eye. Fundus exhibited several nummular perifoveal islands of retinal pigment epithelium atrophy and adjacent pale deposits in both eyes.Retinal anatomy was investigated with autofluorescence, retinal angiography and optical coherence tomography. Retinal function was assessed with automated static perimetry, full-field and multifocal electroretinography and electro-oculography. Genetic testing of mtDNA identified a point mutation at the locus 3243. CONCLUSION: Observation of RPE abnormalities in the context of suggestive systemic findings should prompt mtDNA testing.

Triggering of Bcl-2-related pathway is associated with apoptosis of photoreceptors in Rpe65-/- mouse model of Leber's congenital amaurosis.

Mutations in RPE65 protein is characterized by the loss of photoreceptors, although the molecular pathways triggering retinal cell death remain largely unresolved. The role of the Bcl-2 family of proteins in retinal degeneration is still controversial. However, alteration in Bcl-2-related proteins has been observed in several models of retinal injury. In particular, Bax has been suggested to play a crucial role in apoptotic pathways in murine glaucoma model as well as in retinal detachment-associated cell death. We demonstrated that Bcl-2-related signaling pathway is involved in Rpe65-dependent apoptosis of photoreceptors during development of the disease. Pro-apoptotic Bax alpha and beta isoforms were upregulated in diseased retina. This was associated with a progressive reduction of anti-apoptotic Bcl-2, reflecting imbalanced Bcl-2/Bax ratio as the disease progresses. Moreover, specific translocation of Bax beta from cytosol to mitochondria was observed in Rpe65-deficient retina. This correlated with the initiation of photoreceptor cell loss at 4 months of age, and further increased during disease development. Altogether, these data suggest that Bcl-2-apoptotic pathway plays a crucial role in Leber's congenital amaurosis disease. They further highlight a new regulatory mechanism of Bax-dependent apoptosis based on regulated expression and activation of specific isoforms of this protein.

A novel homozygous R764H mutation in crumbs homolog 1 causes autosomal recessive retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP; MIM 268000) is a hereditary disease characterized by poor night vision and progressive loss of photoreceptors, eventually leading to blindness. This degenerative process primarily affects peripheral vision due to the loss of rods. Autosomal recessive RP (arRP) is clinically and genetically heterogeneous. It has been associated with mutations in different genes, including CRB1 (crumbs homolog 1). The aim of this study was to determine the causative gene in a Tunisian patient with arRP born to non-consanguineous parents. METHODS: Four accessible family members were included. They underwent full ophthalmic examination with best-corrected Snellen visual acuity, fundus photography and fluorescein angiography. Haplotype analysis was used to evaluate homozygosity in the family to 20 arRP loci. All exons and intron-exon junctions of candidate genes not excluded by haplotype analysis were PCR amplified and directly sequenced. RESULTS: The proband was a 43-year-old female patient. Best-corrected visual acuity was 20/63 (right eye) and 20/80 (left eye). Visual loss began during the third decade. Funduscopic examination and fluorescein angiography revealed typical advanced RP changes with bone spicule-like pigment deposits in the posterior pole and the midperiphery along with retinal atrophy, narrowing of the vessels, and waxy optic discs. Haplotype analysis revealed homozygosity with microsatellite markers D1S412 and D1S413 on chromosome 1q31.3. These markers flanked CRB1. Our results excluded linkage of all the other arRP loci/genes tested. Sequencing of the 12 coding exons and splice sites of CRB1 disclosed a homozygous missense mutation in exon 7 at nucleotide c. 2291G>A, resulting in an arginine to histidine substitution (p.R764H). CONCLUSIONS: R764H is a novel mutation associated with CRB1-related arRP. Previously, an R764C mutation was reported. Extending the mutation spectrum of CRB1 with additional families is important for genotype-phenotype correlations and characterization of the scope of mutation.

Comparison of Fundus Autofluorescence Between Fundus Camera (Topcon) and Confocal Scanning Laser Ophthalmoscope (HRA) in Various Pathologies

Purpose: To investigate the differences between the Fundus Camera (Topcon TRC-50X) and Confocal Scanning Laser Ophthalmoscope (Heidelberg retina angiogram (HRA)) on the fundus autofluorescence (FAF) imaging (resolution and FAF characteristics). Methods: Eighty nine eyes of 46 patients with various retinal diseases underwent FAF imaging with HRA (488nm exciter / 500nm barrier filter) before fluorescein angiography (FFA) and Topcon Fundus Camera (580nm exciter / 695nm barrier filter) before and after FFA. The quality of the FAF images was estimated, compared for their resolution and analysed for the influence of fixation stability and cataracts. Hypo- and hyper-FAF behaviour was analysed for the healthy disc, healthy fovea, and a variety of pathological features. Results: HRA images were found to be of superior quality in 18 eyes, while Topcon images were estimated superior in 21 eyes. No difference was found in 50 eyes. Both poor fixation (p=0.009) and more advanced cataract (p=0.013) were found to strongly increase the likelihood of better image quality by Topcon. Images acquired by Topcon before and after FFA were identical (100%). The healthy disc was usually dark on HRA (71%), but showed mild autofluorescence on Topcon (88%). The healthy fovea showed in 100% Hypo-FAF on HRA, while Topcon showed in 52% Iso-FAF, in 43% mild Hypo-FAF, and in 5% Hypo-FAF as on HRA. No difference of FAF was found for geographic atrophy, pigment changes, and drusen, although Topcon images were often more detailed. Hyper-FAF due to exudation showed better on HRA. Pigment epithelium detachment showed identical FAF behaviour on the border, but reduced FAF with Topcon in the center. Cystic edema was visible only on HRA in a petaloid pattern. Hard exsudates caused Hypo-FAF only on HRA, hardly visible on Topcon. Blocage phenomenon by blood however was identical. Conclusions: The filter set of Topcon and the single image acquisition appear to be an advantage for patients with cataract or poor fixation. Preceding FFA does not alter the Topcon FAF image. Regarding the FAF behaviour, there are differences between the two systems which need to be taken into account when interpreting the images.

Four novel Loci (19q13, 6q24, 12q24, and 5q14) influence the microcirculation in vivo.

There is increasing evidence that the microcirculation plays an important role in the pathogenesis of cardiovascular diseases. Changes in retinal vascular caliber reflect early microvascular disease and predict incident cardiovascular events. We performed a genome-wide association study to identify genetic variants associated with retinal vascular caliber. We analyzed data from four population-based discovery cohorts with 15,358 unrelated Caucasian individuals, who are members of the Cohort for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, and replicated findings in four independent Caucasian cohorts (n  =  6,652). All participants had retinal photography and retinal arteriolar and venular caliber measured from computer software. In the discovery cohorts, 179 single nucleotide polymorphisms (SNP) spread across five loci were significantly associated (p<5.0×10(-8)) with retinal venular caliber, but none showed association with arteriolar caliber. Collectively, these five loci explain 1.0%-3.2% of the variation in retinal venular caliber. Four out of these five loci were confirmed in independent replication samples. In the combined analyses, the top SNPs at each locus were: rs2287921 (19q13; p  =  1.61×10(-25), within the RASIP1 locus), rs225717 (6q24; p = 1.25×10(-16), adjacent to the VTA1 and NMBR loci), rs10774625 (12q24; p  =  2.15×10(-13), in the region of ATXN2,SH2B3 and PTPN11 loci), and rs17421627 (5q14; p = 7.32×10(-16), adjacent to the MEF2C locus). In two independent samples, locus 12q24 was also associated with coronary heart disease and hypertension. Our population-based genome-wide association study demonstrates four novel loci associated with retinal venular caliber, an endophenotype of the microcirculation associated with clinical cardiovascular disease. These data provide further insights into the contribution and biological mechanisms of microcirculatory changes that underlie cardiovascular disease.

Protective effect of intravitreal injection of vasoactive intestinal peptide-loaded liposomes on experimental autoimmune uveoretinitis.

PURPOSE: The aim of this study was to investigate the effect of a single intravitreal (i.v.t.) injection of vasoactive intestinal peptide (VIP) loaded in rhodamine-conjugated liposomes (VIP-Rh-Lip) on experimental autoimmune uveoretinitis (EAU). METHODS: An i.v.t. injection of VIP-Rh-Lip, saline, VIP, or empty-(E)-Rh-Lip was performed simultaneously, either 6 or 12 days after footpad immunization with retinal S-antigen in Lewis rats. Clinical and histologic scores were determined. Immunohistochemistry and cytokine quantification by multiplex enzyme-linked immunosorbent assay were performed in ocular tissues. Systemic immune response was determined at day 20 postimmunization by measuring proliferation and cytokine secretion of cells from inguinal lymph nodes (ILNs) draining the immunization site, specific delayed-type hypersensitivity (DTH), and the serum concentration of cytokines. Ocular and systemic biodistribution of VIP-Rh-Lip was studied in normal and EAU rats by immunofluorescence. RESULTS: The i.v.t. injection of VIP-Rh-Lip performed during the afferent, but not the efferent, phase of the disease reduced clinical EAU and protected against retinal damage. No effect was observed after saline, E-Rh-Lip, or VIP injection. VIP-Rh-Lip and VIP were detected in intraocular macrophages and in lymphoid organs. In VIP-Rh-Lip-treated eyes, macrophages expressed transforming growth factor-beta2, low levels of major histocompatibility complex class II, and nitric oxide synthase-2. T-cells showed activated caspase-3 with the preservation of photoreceptors. Intraocular levels of interleukin (IL)-2, interferon-gamma (IFN-gamma), IL-17, IL-4, GRO/KC, and CCL5 were reduced with increased IL-13. At the systemic level, treatment reduced retinal soluble autoantigen lymphocyte proliferation, decreased IL-2, and increased IL-10 in ILN cells, and diminished specific DTH and serum concentration of IL-12 and IFN-gamma. CONCLUSIONS: An i.v.t. injection of VIP-Rh-Lip, performed during the afferent stage of immune response, reduced EAU pathology through the immunomodulation of intraocular macrophages and deviant stimulation of T-cells in ILN. Thus, the encapsulation of VIP within liposomes appears as an effective strategy to deliver VIP into the eye and is an efficient means of the prevention of EAU severity.