Treated Cases of Retinopathy of Prematurity in Germany: 10-Year Data from the Retina.net Retinopathy of Prematurity Registry.

PURPOSE: To analyze changes in demographic parameters and retreatment patterns over a 10-year period in a clinical routine setting of infants with retinopathy of prematurity (ROP) requiring treatment documented in the German Retina.net ROP registry. DESIGN: Multicenter, noninterventional, observational registry study recruiting patients treated for ROP. SUBJECTS: A total of 692 eyes of 353 infants treated for ROP were documented in the Retina.net ROP registry over a 10-year period between 2011 and 2020. These cases cover about 15% of all infants treated for ROP in Germany. METHODS: The Retina.net ROP registry was established in 2012 to jointly collect information on infants treated for ROP. The database collects information on demographic parameters (gestational age [GA], birth weight, neonatal comorbidities) as well as treatment parameters (type of treatment, weight and age at treatment, and stage of ROP). A total of 19 centers contributed to the analysis. This is the 10-year analysis of data from 2011 to 2020, in which we focus on changes over time regarding the respective parameters. MAIN OUTCOME MEASURES: Changes over time in demographic parameters and treatment patterns for ROP in Germany. RESULTS: The overall incidence of treatment requiring ROP was 3.5% of all infants screened for ROP at participating centers. Gestational age, weight at birth, and weight at treatment remained stable over the 10-year period, whereas postmenstrual and postnatal age at treatment increased moderately but statistically significantly over the years. The most prevalent ROP severity stage at treatment was stage 3+ in zone II (76.6% of all treated eyes). Treatment patterns changed considerably from predominantly laser treatments in 2011 (75% of all treated eyes) to predominantly ranibizumab treatments in 2020 (60.9% of all treated eyes). The overall retreatment rate was 15.6%. Retreatment rates differed between initial treatment modalities (14.1% after laser coagulation, 12% after bevacizumab and 24.5% after ranibizumab). Treatment-associated systemic or ophthalmic complications were rare. CONCLUSIONS: This data analysis represents one of the largest documented cohorts of infants treated for ROP. The data on demographic parameters and treatment patterns provide useful information for further improvement of ROP management. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Retinal Vascular Occlusion after COVID-19 Vaccination: More Coincidence than Causal Relationship? Data from a Retrospective Multicentre Study.

BACKGROUND: To investigate whether vaccination against SARS-CoV-2 is associated with the onset of retinal vascular occlusive disease (RVOD). METHODS: In this multicentre study, data from patients with central and branch retinal vein occlusion (CRVO and BRVO), central and branch retinal artery occlusion (CRAO and BRAO), and anterior ischaemic optic neuropathy (AION) were retrospectively collected during a 2-month index period (1 June-31 July 2021) according to a defined protocol. The relation to any previous vaccination was documented for the consecutive case series. Numbers of RVOD and COVID-19 vaccination were investigated in a case-by-case analysis. A case-control study using age- and sex-matched controls from the general population (study participants from the Gutenberg Health Study) and an adjusted conditional logistic regression analysis was conducted. RESULTS: Four hundred and twenty-one subjects presenting during the index period (61 days) were enrolled: one hundred and twenty-one patients with CRVO, seventy-five with BRVO, fifty-six with CRAO, sixty-five with BRAO, and one hundred and four with AION. Three hundred and thirty-two (78.9%) patients had been vaccinated before the onset of RVOD. The vaccines given were BNT162b2/BioNTech/Pfizer (n = 221), followed by ChadOx1/AstraZeneca (n = 57), mRNA-1273/Moderna (n = 21), and Ad26.COV2.S/Johnson & Johnson (n = 11; unknown n = 22). Our case-control analysis integrating population-based data from the GHS yielded no evidence of an increased risk after COVID-19 vaccination (OR = 0.93; 95% CI: 0.60-1.45, p = 0.75) in connection with a vaccination within a 4-week window. CONCLUSIONS: To date, there has been no evidence of any association between SARS-CoV-2 vaccination and a higher RVOD risk.

High-Throughput Sequencing to Identify Mutations Associated with Retinal Dystrophies.

Retinal dystrophies (RD) are clinically and genetically heterogenous disorders showing mutations in over 270 disease-associated genes. Several millions of people worldwide are affected with different types of RD. Studying the relevance of disease-associated sequence alterations will assist in understanding disorders and may lead to the development of therapeutic approaches. Here, we established a whole exome sequencing (WES) pipeline to rapidly identify disease-associated mutations in patients. Sanger sequencing was applied to identify deep-intronic variants and to verify the co-segregation of WES results within families. We analyzed 26 unrelated patients with different syndromic and non-syndromic clinical manifestations of RD. All patients underwent ophthalmic examinations. We identified nine novel disease-associated sequence variants among 37 variants identified in total. The sequence variants located to 17 different genes. Interestingly, two cases presenting with Stargardt disease carried deep-intronic variants in ABCA4. We have classified 21 variants as pathogenic variants, 4 as benign/likely benign variants, and 12 as variants of uncertain significance. This study highlights the importance of WES-based mutation analyses in RD patients supporting clinical decisions, broadly based genetic diagnosis and support genetic counselling. It is essential for any genetic therapy to expand the mutation spectrum, understand the genes' function, and correlate phenotypes with genotypes.

Comparing Alternative Ranibizumab Dosages for Safety and Efficacy in Retinopathy of Prematurity: A Randomized Clinical Trial.

Importance: Anti-vascular endothelial growth factor (VEGF) therapies are a novel treatment option in retinopathy of prematurity (ROP). Data on dosing, efficacy, and safety are insufficient. Objective: To investigate lower doses of anti-VEGF therapy with ranibizumab, a substance with a significantly shorter systemic half-life than the standard treatment, bevacizumab. Design, Setting, and Participants: This randomized, multicenter, double-blind, investigator-initiated trial at 9 academic medical centers in Germany compared ranibizumab doses of 0.12 mg vs 0.20 mg in infants with bilateral aggressive posterior ROP; ROP stage 1 with plus disease, 2 with plus disease, or 3 with or without plus disease in zone I; or ROP stage 3 with plus disease in posterior zone II. Patients were recruited between September 2014 and August 2016. Twenty infants were screened and 19 were randomized. Interventions: All infants received 1 baseline ranibizumab injection per eye. Reinjections were allowed in case of ROP recurrence after at least 28 days. Main Outcomes and Measures: The primary end point was the number of infants who did not require rescue therapy at 24 weeks. Key secondary end points included time-to-event analyses, progression of physiologic vascularization, and plasma VEGF levels. Stages of ROP were photodocumented and reviewed by an expert committee. Results: Nineteen infants with ROP were enrolled (9 [47.4%] female; median [range] postmenstrual age at first treatment, 36.4 [34.7-39.7] weeks), 3 of whom died during the study (1 in the 0.12-mg group and 2 in the 0.20-mg group). Of the surviving infants, 8 (88.9%) (17 eyes [94.4%]) in the 0.12-mg group and 6 (85.7%) (13 eyes [92.9%]) in the 0.20-mg group did not require rescue therapy. Both ranibizumab doses were equally successful in controlling acute ROP (Cochran-Mantel-Haenszel analysis; odds ratio, 1.88; 95% CI, 0.26-13.49; P = .53). Physiologic intraretinal vascularization was superior in the 0.12-mg group. The VEGF plasma levels were not systematically altered in either group. Conclusions and Relevance: This pilot study demonstrates that ranibizumab is effective in controlling acute ROP and that 24% of the standard adult dose (0.12 mg) appears equally effective as 40% (0.20 mg). Superior vascularization of the peripheral retina with 0.12 mg of ranibizumab indicates that the lower dose may be favorable. Unchanged plasma VEGF levels point toward a limited systemic drug exposure after ranibizumab. Trial Registration: clinicaltrials.gov Identifier: NCT02134457 and clinicaltrialsregister.eu Identifier: 2013-002539-13.

Primary vitrectomy for rhegmatogenous retinal detachment in pseudophakic eyes: 20-gauge versus 25-gauge vitrectomy.

PURPOSE: To report anatomical and functional outcome of 20-gauge versus 25-gauge primary pars plana vitrectomy for management of complex rhegmatogenous retinal detachment in pseudophakic eyes. METHODS: Prospective single-centre randomized comparative pilot trial. Fifty patients with retinal detachment (RD) not complicated by proliferative vitreoretinopathy grade B or C, who cannot be treated with a single meridional sponge, were randomized (1:1) from November 2006 to January 2010 to either 20-gauge or 25-gauge vitrectomy as first surgical intervention and followed up over a 12-month period, evaluating change in best-corrected visual acuity, anatomical success and intraocular pressure dysregulation. RESULTS: Mean visual acuity improved by 0.88 (SD 0.67) from 1.22 logMAR (SD 0.63) to 0.34 logMAR (SD 0.31) in the 20-gauge group and by 0.53 (SD 0.91) from 0.86 logMAR (SD 0.73) to 0.34 logMAR (SD 0.46) in the 25-gauge group. Final anatomical success rate was 100% and primary success rate was 69% at 6 months of follow-up. In the 20-gauge group, the retina was attached after one single procedure in 18 eyes (72%) and in 21 eyes (84%) of the 25-gauge group. Two patients in the 25-gauge group had hypotony at the first postoperative day which normalized within 6 weeks. CONCLUSION: In our series, transconjunctival sutureless 25-gauge and 20-gauge vitrectomy showed comparable results in pseudophakic RD not suitable for single sponge surgery with respect to visual outcome and retinal reattachment. Postoperative hypotony does not seem to be a significant problem of transconjunctival sutureless vitrectomy.

The German ROP Registry: data from 90 infants treated for retinopathy of prematurity.

PURPOSE: The German retinopathy of prematurity (ROP) Registry collects data on treated ROP in a multicentre approach to analyse epidemiology and treatment patterns of severe ROP. METHODS: Nine centres entered data from 90 treated ROP infants (born between January 2011 and December 2013) into a central database. Analysis included incidence rate of severe ROP, demographic data, stage of ROP, treatment patterns, recurrence rates, relevant comorbidities and ophthalmological or systemic complications associated with treatment. RESULTS: Treatment rate for ROP was 3.2% of the screened population. The most frequent ROP stage at time of treatment was zone II, stage 3 +  (137 eyes). Treatment was bilateral in 97% of infants. Treatment patterns changed over time from 7% anti-vascular endothelial growth factor (VEGF) monotherapy in 2011 to 32% in 2014. Overall, laser treatment was the predominant treatment. However, all infants with zone I disease received anti-VEGF treatment. About 19% of infants required retreatment (16% of laser-treated and 21% of anti-VEGF treated infants). Mean time between first and second treatment was 3.8 weeks (± 11 days) for laser-treated and 10.4 weeks (± 60 days) for anti-VEGF-treated infants. CONCLUSION: This study is the first multicentre analysis of severe ROP in Germany. The identified treatment patterns find laser as the most prevalent form of therapy, with an increasing use of anti-VEGF therapy over recent years. Recurrence rates were relatively high overall with slightly higher recurrence rates and later recurrence times in the anti-VEGF group. Anti-VEGF was predominantly used for high-risk stages like AP-ROP and zone I disease.

Toxic effects of melphalan, topotecan and carboplatin on retinal pigment epithelial cells.

PURPOSE: Clinical evidence of retinal pigment epithelium (RPE) alterations after intra-arterial (IAC) and intravitreal chemotherapy (IViC) of retinoblastoma has been reported. We, therefore, investigated the cellular toxic effects of melphalan, topotecan and carboplatin on the RPE in a cell culture model. METHODS: The effects of melphalan, carboplatin and topotecan on ARPE19 cell morphology were examined by phase contrast microscopy. Cell proliferation was quantified by BrdU incorporation, cell viability studied via MTS assays, and cell densities were estimated by Crystal Violet staining, and apoptosis induction studied via caspase 3/7-activity assays after a 24-hr incubation period. Staurosporine, media without fetal bovine serum, diluents of melphalan, carboplatin and topotecan were applied as positive and negative controls, respectively. RESULTS: We observed a concentration-dependent increase in the number and size of gaps in the ARPE19 cell layer with each drug. There was a significant decrease in proliferative activity and cell viability of RPE cells as well as an increase in apoptosis after 24 hrs culture in media supplemented with melphalan and topotecan. Carboplatin had comparable effects on cell proliferation and cell viability; however, no significant apoptotic impacts were observed. The three cytostatic drugs had insignificant effects on cell density measurements. CONCLUSIONS: Morphological monitoring and toxicity assays indicate a direct toxic effect of melphalan and the other two cytostatic drugs on ARPE19 cells. Thus, a direct toxic effect of melphalan in vivo after IAC or IViC on the RPE seems probable and may explain the clinical and angiographic RPE alterations observed in some retinoblastoma patients.

Investigating short-term toxicity of melphalan in a model of an isolated and superfused bovine retina.

BACKGROUND: Melphalan, as a treatment for retinoblastoma, has been applied intra-arterially by catheterisation of the ophthalmic artery or intravitreally, aiming to reduce systemic side effects of intravenous drug therapy. This study evaluates retinal toxicity of different melphalan concentrations measured by electroretinogram (ERG) in an isolated and perfused retinal whole mount culture. METHODS: For functional testing, bovine retinas were prepared and perfused with an oxygen-saturated standard solution and the ERG was recorded until stable b-wave or a-wave amplitudes were reached. Thereafter, retinae were exposed to 80, 160 and 320 µg/ml of melphalan for 30 min. After exposure, a washout was performed thrice for 5 min each and the ERG amplitude recovery was monitored for 60 min. To investigate the effects on photoreceptor function, 1-mM asparate was added to suppress the b-wave and obtain isolated a-waves. RESULTS: While no toxic effects for a concentration of 80 µg/ml were observed, both b- and a-waves were significantly reduced after application of 160 (b-wave 43.8 %, p = 0.03; a-wave 28.2 %, p = 0.04) and 320 µg/ml (b-wave 20.0 %, p = 0.04; a-wave 35.8 %, p = 0.02). For 320 µg/ml, this reduction remained significant at the end of the washout (b-wave 40.0 % p = 0.02; a-wave 26.4 %, p = 0.02). CONCLUSIONS: Epiretinal or intraretinal concentrations of 80-µg/ml melphalan do not cause toxic effects in this in vitro model. Concentrations higher than 160 µg/ml should be avoided.

Electrophysiological toxicity testing of VEGF Trap-Eye in an isolated perfused vertebrate retina organ culture model.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of visual impairment in Western nations. Since the discovery of the importance of vascular endothelial growth factor (VEGF) in the pathogenesis of neovascular AMD, anti-VEGF agents including pegaptanib, ranibizumab and bevacizumab provide a treatment option to improve vision in affected persons. VEGF Trap-Eye (Aflibercept) is a new agent available for the treatment of exudative AMD. The molecule is a receptor decoy with a longer half-life and a higher affinity to VEGF compared with ranibizumab or bevacizumab. The presented study has been designed to evaluate the short-term toxic effects of VEGF Trap-Eye on retinal function during and after direct exposure to the drug. METHODS: Isolated bovine retinas were perfused with an oxygen-saturated nutrient solution, and the electroretinogram (ERG) was recorded using silver/silver chloride electrodes. A total of 0.5 mg or 2 mg VEGF Trap-Eye was added to the nutrient solution and retinas were exposed for 45 min, followed by a washout period of 100 min. The percentage of a- and b-wave reduction at the end of the washout was compared with the baseline values. Additionally, retinal whole mount cultures were exposed for 24 hr to VEGF Trap-Eye, and the amount of apoptotic cells were determined using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labelling (TUNEL) assay. RESULTS: During simulation of intraocular application, no significant reduction in the a-wave amplitude for 0.5 mg (2.70%, p = 0.37) and 2 mg (3.84%, p = 0.37) VEGF Trap-Eye and b-wave amplitude for 0.5 mg (19.68%, p = 0.17) and 2 mg (24.1%, p = 0.06) VEGF Trap-Eye was observed at the end of the washout. However, there were significant changes in a-wave and b-wave amplitudes directly after exposure to 0.5 mg VEGF Trap-Eye (18.4%, p = 0.004 and 43.1%, p = 0.006, respectively). CONCLUSIONS: The presented data suggest that intraocular application of up to 2 mg VEGF Trap-Eye does not induce irreversible toxic retinal damage. However, short-term results showed a negative effect directly after the application for 0.5 mg and 2 mg VEGF Trap-Eye.

Pars plana vitrectomy for treatment of advanced Coats' disease--presentation of a modified surgical technique and long-term follow-up.

BACKGROUND: To present a modified surgical technique in the treatment of retinal detachment secondary to advanced Coats' disease in children, and report on long-term anatomical and functional outcome. METHODS: We analysed an interventional case series of 13 patients (13 eyes) with advanced Coats' disease characterised by retinal detachment in addition to massive subretinal exudates and vascular malformation. The presented patients underwent pars plana vitrectomy (PPV), including a modified technique of exocryotherapy applied after fluid-air exchange in order to achieve complete treatment of the vascular changes, to reduce associated side-effects, and to avoid retinectomy and silicone oil tamponade. RESULTS: Within a median follow-up period of 37 months (range: 18-66 months), no enucleation was necessary. Four eyes (31 %) did not need any further therapy, and in nine eyes (69 %) additional treatments were performed. Six patients (46 %) required revisional surgery with silicone oil tamponade. In ten eyes (77 %), the pathologic vessels and exudates finally regressed and the retina reattached. Visual acuity (VA) could be stabilized in the majority of patients: in three eyes (27 %) VA improved, in four eyes (36 %) VA remained stable, in four eyes (36 %) visual acuity (VA) deteriorated, and in two eyes VA could not be evaluated. CONCLUSIONS: The presented modified technique allows for sufficient cryotherapy of vascular malformations, even in the presence of massive exudation, in a subset of patients with advanced Coats' disease, and thus may reduce surgery-related complications and improve the rehabilitation process of these young patients.

Cataract formation: a possible complication of intra-arterial chemotherapy for retinoblastoma.

PURPOSE: To delineate and discuss a not yet described possible ocular complication of selective intra-arterial chemotherapy (SIAC) for treatment of retinoblastoma. METHODS: A 23-month-old girl with a large unilateral retinoblastoma was treated with repeated SIAC using 5 mg melphalan between July 2010 and January 2012. Clinical course of tumor and further ocular changes after therapy and histopathologic findings are described. RESULTS: In total, 5 SIAC were performed over a time period of 18 months. After the last SIAC, diffuse dense cataract prevented further funduscopy. In addition, anterior chamber seeding was obvious, leading to the decision to enucleate the eye. Histopathologically, nearly complete regression of the main tumor mass with prominent calcifications, but vital tumor seeding in the vitreous, on the lens surface, on the ciliary body, and in the anterior chamber, was observed. Peculiar vacuolation of the lens epithelial cells, liquefaction of the subepithelial lens fibers, and diffuse small vacuoles within the lens were striking. CONCLUSIONS: Repeated SIAC with melphalan may induce cataract formation, possibly as a toxic effect of the chemotherapeutic to the lens, maybe combined with radiation exposure during fluoroscopy. This ocular complication should be taken into consideration as a limitation of the number of feasible repeated treatments.

Comparative toxicity and proliferation testing of aflibercept, bevacizumab and ranibizumab on different ocular cells.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is a key factor in the pathogenesis of neovascular retinal diseases including age-related macular degeneration. VEGF inhibitors including ranibizumab, pegaptanib or bevacizumab improve retinal morphology and vision in many patients. The recently approved drug aflibercept (VEGF Trap-Eye/Eyelea, Regeneron, Tarrytown, New York, USA) offers a new therapy modality. We therefore tested for toxic and anti-proliferating effects of aflibercept. METHODS: The effects of aflibercept (0.125, 0.5, 2 mg), ranibizumab (0.125 mg) and bevacizumab (0.3125 mg) after 1, 24, 48 and 72 h on cell morphology via phase contrast pictures, cell viability via MTS assay, total cell amount via crystal violet staining, apoptosis induction via caspase 3/7 assay and proliferation via BrdU assay were investigated. Three ocular cell lines were chosen for toxicology testing: ARPE19 cells, RGC-5 cells and 661W cells. RESULTS: Aflibercept did not cause changes in cell morphology, induce apoptosis or cause permanent decrease in cell viability, cell density or proliferation in any cell line or concentration investigated. In general, aflibercept had fewer effects (upregulation or downregulation) compared with controls than bevacizumab or ranibizumab. CONCLUSIONS: In our experiments, aflibercept did not lead to any negative effects on retinal cell lines and might therefore be used safely in clinical applications.

Amniotic bands as a cause of congenital anterior staphyloma.

BACKGROUND: Congenital anterior staphyloma is a rare, complex malformation syndrome of the anterior segment. Only a few reports on associated systemic malformations have been published. We herein present a rare manifestation of congenital anterior staphyloma (CAS) combined with amniotic band disruption syndrome (ABS). PATIENT AND METHODS: Shortly after birth, a massive enlargement of the left eye was observed in a female child. Furthermore, an extensive bilateral congenital cleft lip and cleft alveolar ridge with oblique facial cleft extending into the left medial canthal region, coloboma(s) of the left eyelids, extensive adhesions between lids and eye bulb, as well as circumferential grooves, clubfeet, and terminal transverse defects in both hands and feet were present. Due to severe progression of eye bulb protrusion with thinning of the sclera, enucleation of the left eye was performed at the age of 3 years in order to prevent complications including perforation of the globe and with the aim of improving cosmetic aspects. RESULTS: Histopathological examination of the enucleated eye disclosed findings typical of congenital anterior staphyloma, including massive corneal staphylomatic deformation with superficial vascularization and elapsed corneoscleral margin, destruction of Bowman's layer, absence of Descemet's layer, corneal endothelium, and angle structures. The lens was only partially formed, and had mainly dissolved. The neural retina appeared normal. The optic nerve disc revealed a pronounced excavation. Facial clefts, lid colobomas, congenital constriction bands, and amputation of distal limbs match ABS. This malformation complex develops in early pregnancy, probably prior to 35 days post conception. CONCLUSION: This is the first report on an association of these two rare complex congenital malformations, congenital anterior staphyloma and amniotic band syndrome. The anterior staphyloma was unilateral, and related to facial clefts and lid coloboma in the area adjacent to the anterior staphyloma. Furthermore, the systemic deformities are clearly due to the amniotic bands, and the timing of the development of both complex malformations seems to be similar. All findings suggest that the presence of amniotic bands is a causative factor for all observed abnormalities including anterior staphyloma.

Identification of a novel CRYBB2 missense mutation causing congenital autosomal dominant cataract.

PURPOSE: To identify the genetic defect in a four-generation Croatian family presenting with autosomal dominant cataract. METHODS: Genome-wide linkage analysis with 250K single nucleotide polymorphism (SNP) arrays was performed using DNA from one unaffected and seven affected individuals. Mutation screening of candidate genes was performed by bidirectional Sanger sequencing. RESULTS: Evidence for linkage was observed for eight genomic regions. Among these was a locus on chromosome 22 which encompasses the ß-crystallin gene cluster. This cluster includes four genes, namely beta-crystallin B1 (CRYBB1), beta-crystallin B2 (CRYBB2), beta-crystallin B3 (CRYBB3), and beta-crystallin A4 (CRYBA4). A novel sequence variant was found in the CRYBB2 gene (p.Arg188His). This variant cosegregated with the disease phenotype in all affected individuals but was not present in the unaffected family members and 100 healthy control subjects. CONCLUSIONS: We report a novel missense mutation, p.Arg188His, in CRYBB2 associated with congenital cataract in a family of Croatian origin. This variant is the most COOH-terminal missense mutation in CRYBB2 that has been identified so far.

Non-arteritic anterior ischemic optic neuropathy (NA-AION) after intravitreal injection of bevacizumab (Avastin) for treatment of angoid streaks in pseudoxanthoma elasticum.

BACKGROUND: To report a case of nonarteritic anterior ischemic optic neuropathy (NA-AION) following intravitreal injection of bevacizumab (Avastin). METHODS: Interventional case report with an 18-month follow-up. RESULTS: A 51-year-old male with pseudoxanthoma elasticum presented with NA-AION 2 weeks after treatment with intravitreal of bevazicumab (Avastin) for choroidal neovascularisation secondary to angioid streaks. Except from a small optic disc without cupping he did not show further risk factors. DISCUSSION: Risk of NA-AION should be taken into consideration when deciding for intravitreal application of drugs including anti-vascular endothelial growth factors (VEGF) agents like bevacizumab (Avastin) in the treatment of retinal vascular diseases.

Analysis of the neuronal marker protein gene product 9.5 in internal limiting membranes after indocyanine-green assisted peeling.

BACKGROUND: Indocyanine green-assisted internal limiting membrane (ILM) peeling was suspected to disrupt the innermost layer of the neural retina. We examined whether surgically excised specimens contain remnants of neuronal tissue. METHODS: Ten patients with macular hole underwent pars plana vitrectomy and indocyanine green-assisted ILM peeling. A total of 0.1 mL of a 0.5% indocyanine green solution was applied for 15 seconds. The ILM specimens were prepared for immunohistochemistry, using a polyclonal antibody against protein gene product 9.5. Protein gene product 9.5 is a pan-neuronal marker labeling human neuronal cells. Appropriate controls to show selectivity of the antibody were performed on neuronal tissue of donor eyes. One ILM was prepared for electron microscopy. RESULTS: A selective expression of protein gene product 9.5 was found in neuronal fibers of the retina and optic nerve of donor eyes. Only 1 of the 10 surgical ILM specimens showed a minimal focal positivity for protein gene product 9.5. No neuronal tissue was detected on the ILM by electron microscopy. CONCLUSION: Focal expression of protein gene product 9.5 in only 1 of 10 surgical ILM specimens argues against a general indocyanine green-related disruption of the innermost retinal layers. However, higher concentrations of the dye, longer incubation times or different solvents than used in this study may lead to different results.

Sodium hyaluronate gels as a drug-release system for corticosteroids: release kinetics and antiproliferative potential for glaucoma surgery.

PURPOSE: To evaluate the release kinetics, biocompatibility and antiproliferative potential of a concentrated hydrophilic steroid formulation from commercially available sodium hyaluronate gels as a potential adjunct in glaucoma surgery. METHODS: Dexamethasone and sodium hyaluronate 1% (Healon) and sodium hyaluronate 2.3% (Healon 5) were mixed to yield sodium hyaluronate formulations containing dexamethasone in concentrations of 4-20 mg/ml (7.7-38 mm). Non-cumulative and cumulative release into balanced salt solution (BSS) or phosphate buffered saline (PBS) was measured spectrophotometrically over 2-6 days. For cytotoxicity assays, human tenon fibroblasts (HTFB) and human retinal pigment epithelium cells (ARPE19) were cultured in a serum-deficient medium to ensure a static milieu; 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay and Live/Dead cell-mediated cytotoxicity assay were used to exclude cytotoxicity. Cellular proliferative activity was monitored by 5'-bromo-2'-deoxyuridine (BrdU)-incorporation into cellular DNA. RESULTS: The release kinetics from sodium hyaluronate 1% and 2.3% were almost identical. Steady state was achieved after approximately 44 hrs in non-cumulative measurements. The release plotted as a function of the square root of time was consistent with a largely diffusion-controlled release system. No cytotoxicity could be observed. Dexamethasone-loaded sodium hyaluronate showed a significant antiproliferative effect on HTFB and ARPE19 cells. CONCLUSION: Dexamethasone-loaded sodium hyaluronate shows extended release of steroid over almost 2 days in concentrations high enough to inhibit the proliferation of HTFB and RPE cells without evoking cytotoxic effects. Thus, this formulation may be an easy-to-prepare adjunct in glaucoma surgery or other procedures in which cellular growth inhibition is desired.

Vitreous levels of bevacizumab and vascular endothelial growth factor-A in patients with choroidal neovascularization.

PURPOSE: To investigate the vitreous levels of bevacizumab and vascular endothelial growth factor-A (VEGF-A) after intravitreal injection of the drug in patients with choroidal neovascularization (CNV). DESIGN: Interventional case series. PARTICIPANTS: Eleven eyes of 11 patients with submacular hemorrhage and CNV due to age-related macular degeneration (n = 10) or angioid streaks (n = 1). METHODS: All patients were treatment naïve except for a single dose of intravitreal injection of bevacizumab (1.25 mg/50 muL dose) and subsequent vitrectomy after various intervals (1-101 days) because of active and progressive lesion. Intravitreal free bevacizumab and VEGF-A levels were measured using enzyme-linked immunosorbent assay and microsphere-based immunoassay, respectively. Vitreous VEGF-A isoforms were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting. MAIN OUTCOME MEASURES: Intravitreal bevacizumab and VEGF-A levels were measured and pharmacokinetic parameters were calculated. RESULTS: Pharmacokinetics of intravitreal bevacizumab followed a 2-compartment model with initial and terminal half-lives of 0.5 and 6.7 days, respectively. Bevacizumab could be detected in all cases, ranging from 2.63 ng/ml to 165 microg/ml. The peak concentration was observed on the second day after intravitreal bevacizumab injection. Vitreous free VEGF-A levels ranged from 0.2 to 33.9 pg/ml and showed a negative correlation with the bevacizumab concentration (P<0.001; r = -0.955) and a positive correlation with time (P<0.001; r = 0.964). However, the percentage expression of VEGF-A(165) exhibited a positive correlation with the bevacizumab concentration (P = 0.032, r = 0.645) and a negative correlation with time (P = 0.007, r = -0.755). A time-dependent increase was found for the percentage expression of VEGF-A(189) (P = 0.023, r = 0.673). Neither bevacizumab- nor time-related alterations were found for VEGF-A(121). CONCLUSIONS: Based on pharmacokinetics, the interval of 6-7 weeks would be appropriate for efficacy, although clinical trials should guide dosing recommendations. Vitreous levels of free VEGF-A showed a negative correlation with the bevacizumab concentration, which confirmed the in vivo binding affinity of bevacizumab to VEGF-A. The analysis of the VEGF-A isoforms suggests differences of interaction between bevacizumab and individual VEGF-A isoforms.