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.

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.

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.

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.

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.

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.

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.

Efficacy of intravitreal bevacizumab in treating postoperative pseudophakic cystoid macular edema.

PURPOSE: To describe the efficacy of intravitreal bevacizumab (Avastin) in patients with cystoid macular edema (CME) after cataract surgery (Irvine-Gass syndrome). METHODS: This retrospective case series comprised 16 eyes of 16 patients with CME after cataract surgery refractory to current standard treatment who received an injection of 1.25 mg intravitreal Avastin. The main outcome measures were visual acuity, retinal thickness on optical coherence tomography (OCT), and complications related to treatment. RESULTS: The median duration of CME before treatment with intravitreal Avastin was 14 weeks (range 3 to 84 weeks). Although the mean retinal thickness decreased slightly after intravitreal Avastin, the mean visual acuity remained unchanged. Visual acuity improved by 2 Early Treatment Diabetic Retinopathy Study lines in 1 patient, remained unchanged in 12 patients, and decreased by 2 lines in 2 patients. Repeated Avastin injections did not result in a better outcome. Other than mild ocular irritation, there were no adverse effects of the intravitreal injections. CONCLUSIONS: Intravitreal injection of Avastin, although safe, did not result in improved visual function in patients with postoperative CME. In contrast to findings in a previous case report, the beneficial effect of vascular endothelial growth factor inhibition in Irvine-Gass syndrome was not observed.

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.

Long-term follow-up of macular translocation with 360 degrees retinotomy for exudative age-related macular degeneration.

OBJECTIVE: To assess long-term functional and morphological changes after macular translocation in patients with exudative age-related macular degeneration. METHODS: Evaluation of a noncomparative cohort study of 90 patients with a follow-up of 14 to 79 months (mean, 38.2 months). RESULTS: Visual acuity increased by 3 or more lines in 15 patients, remained stable in 35 patients, and deteriorated in 40 patients at final examination. Pigment epithelium atrophy extending to the new fovea was detected in 44 patients; in 25 patients this new atrophy was associated with loss of visual function. CONCLUSIONS: Long-term follow-up of macular translocation with 360 degrees retinotomy showed stabilization or improvement in half of the patients. Progressive atrophy of the pigment epithelium represented one major limiting factor of the beneficial effect of the treatment. Macular translocation may be an option for cases of exudative age-related macular degeneration that are not eligible for or do not respond to alternative treatments.

Safety profile of bevacizumab on cultured human corneal cells.

PURPOSE: To study the corneal biocompatibility of bevacizumab on various cultured human corneal cells. METHODS: Cell cultures of corneal keratinocytes (CKs), corneal fibroblasts (CFs), and corneal endothelial cells (CECs) were harvested from human donor eyes and exposed to various concentrations of bevacizumab (0.25-5.0 mg/mL). Cell viability was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at days 1 and 4 after exposure. For cytotoxicity testing, confluent cells were cultured in serum-depleted medium, and the MTT test was performed after 24 hours of incubation. Expression of vascular endothelial growth factor (VEGF), VEGF receptors (VEGFR1 and VEGFR2), keratan sulphate (KS), and cytokeratin-3 (AE5) was studied by immunohistochemistry. Live/dead viability/cytotoxicity assay was performed and analyzed by fluorescence microscopy after 24 hours of incubation. Cell morphology was assessed with a phase-contrast microscope after 7 days of exposure with different concentrations of bevacizumab (0.25-5.0 mg/mL), and signs of cellular damage were assessed. RESULTS: No cytotoxic effect of bevacizumab on CKs, CFs, and CECs could be observed when used at a concentration of 5.0 mg/mL or lower. Bevacizumab-treated cells showed no signs of cellular damage compared with the control. CKs, CFs, and CECs stained positively for VEGF, VEGFR1, and VEGFR2. CKs and CECs stained positively for AE5, whereas CFs were immunopositive for KS. CONCLUSIONS: Bevacizumab is not toxic to corneal cells of human origin in vitro at doses usually used for treatment of corneal neovascularization, which is 20-fold higher than that used for intravitreal application.

Retinal pigment epithelial cells synthesize laminins, including laminin 5, and adhere to them through alpha3- and alpha6-containing integrins.

PURPOSE: Retinal diseases are often accompanied by changes in the structure of the multilayered extracellular matrix underlying the retina, Bruch's membrane (BrM). These structural revisions potentially lead to alterations in retinal pigment epithelium (RPE) adhesion, likely via modification of interactions with extracellular matrix (ECM) proteins including laminins in BrM. The purpose of this study was to identify specific laminins in BrM and their receptors in RPE cells. METHODS: The laminin composition of BrM was determined using biochemical, molecular biological, and immunohistochemical techniques of rat, bovine, and human tissue and cell lines. An adhesion assay was used to test RPE attachment to laminins and the receptors used for this attachment. RESULTS: BrM contained laminin chains that could form laminin heterotrimers including laminins 1, 5, 10, and 11. RPE cells synthesized these laminin chains in vitro. Therefore, RPE cells may synthesize BrM laminins. The RPE cells preferentially adhered to potential BrM laminins. Although the cells adhered to the BrM component collagen IV, these cells preferentially adhered to laminins. Of the laminins tested, the RPE cells adhered preferentially to laminin 5. The cells interacted with these laminins via specific integrins and attained a different morphology on each laminin. In particular, the RPE cells rapidly attached and flattened on laminin 5. CONCLUSIONS: BrM contains specific laminins, and RPE cells express integrin receptors for those laminins. The interaction of these specific laminins and integrins most likely leads to differential behavior of RPE cells.

Ultrastructural changes after artificial retinal detachment with modified retinal adhesion.

PURPOSE: Artificial retinal detachment is increasingly used in submacular surgery. However, overcoming physiological retinal adhesiveness by subretinal fluid injection is suspected to cause cellular damage and thus to limit visual rehabilitation. This experimental study was designed to examine the ultrastructural changes induced by retinal detachment under vitrectomy conditions and to evaluate factors that reduce adhesiveness and minimize cellular damage. METHODS: Twenty-one pigmented rabbits underwent vitrectomy, and the vitreous cavity was perfused for 10 minutes with various solutions. These included variations in osmolarity (314 and 500 mOsM), Ca(2+) ion concentration (Ca(2+)-supplemented, low Ca(2+), active Ca(2+) deprivation via 1 mM EDTA), temperature (19 degrees C and 34 degrees C), and ischemia (5 minutes). Nonvitrectomized eyes served as the control. Consecutively, an artificial bleb detachment was created underneath the visual streak by injecting 1 mL of buffered saline solution subretinally. Eyes were enucleated within 3 minutes, fixed with 2% glutaraldehyde/0.1 M cacodylate buffer (pH 7.4) containing 100 mM sucrose and processed for transmission electron microscopy and scanning electron microscopy. RESULTS: If a Ca(2+)-containing standard solution was used during vitrectomy, retinal adhesiveness was strong, and a forced bleb detachment caused substantial cellular damage characterized by swollen and fragmented photoreceptor outer segments and disruption of retinal pigment epithelial cells. Use of a Ca(2+)-free solution moderately reduced the adhesive strength with consequently less ultrastructural damage. Active Ca(2+)-deprivation further reduced the retinal adhesion, but may have induced damage as suggested by intracellular vacuolization. Hyperosmolarity and ischemic conditions had toxic effects on both the photoreceptors and RPE cells. In contrast, the use of a preheated Ca(2+)-free solution (34 degrees C) substantially reduced retinal adhesiveness under vitrectomy conditions and hence ultrastructural damage. CONCLUSIONS: Artificial retinal detachment causes substantial ultrastructural damage in eyes with physiological retinal adhesiveness if performed under vitrectomy conditions similar to surgery in humans. The use of a preheated Ca(2+)-free physiologic saline solution seems to be suitable to reduce retinal adhesion sufficiently, without causing significant cellular damage.

Iris pigment epithelial translocation in the treatment of exudative macular degeneration: a 3-year follow-up.

OBJECTIVE: To report the functional and anatomical outcome of 20 patients who underwent surgical removal of choroidal neovascularization combined with transplantation of autologous iris pigment epithelial cells to the subretinal space 3 years after treatment. METHODS: Freshly isolated autologous iris pigment epithelial cells were translocated to the subretinal space in 20 patients after membrane extraction. Patients were followed up by funduscopy, angiography, microperimetry, and visual acuity testing. RESULTS: After a follow-up of 3 years, 1 patient showed improved visual acuity, 13 patients retained stable visual acuity, and 3 patients had reduced visual acuity. No macular edema or recurrent choroidal neovascularization was apparent at any time during the follow-up. CONCLUSIONS: Transplanted autologous iris pigment epithelial cells were well tolerated for 3 years and stabilization of visual acuity was achieved in most patients. These results suggest that iris pigment epithelial cells may serve as a substitute for retinal pigment epithelial cells after choroidal neovascularization removal in patients with exudative macular degeneration; however, whether these cells will be of any value for the restoration of vision and possible protection against choroidal neovascularization recurrence awaits further clinical observation and additional research.

Sutureless amniotic membrane fixation using fibrin glue for ocular surface reconstruction in a rabbit model.

PURPOSE: Amniotic membrane transplantation has become an important treatment option for corneal surface reconstruction. However, suture fixation of the transplant has various disadvantages like corneal irritation, scarring, graft loss due to membrane shrinkage, and the need for subsequent suture removal. Replacement of sutures by bioadhesives might be an advantageous alternative. This controlled study was designed to evaluate a new sutureless technique for amniotic membrane fixation onto the corneal surface by using fibrin glue. METHODS: Standardized disks of cryopreserved amniotic membranes were transplanted onto the deepithelialized cornea of 12 rabbits using either conventional suture fixation or a new fibrin glue technique. The rabbits were followed-up with slit-lamp examination and fluorescein staining until epithelialization was completed. Consecutively, the rabbits were killed and the eyes processed for histology and immunohistochemistry for cytokeratin-3. RESULTS: All membranes of both groups stayed in place throughout the follow-up time and showed a progressive graft epithelialization that was completed after 12 days. Whereas suture-fixated membranes showed progressive tissue shrinkage, fibrin-glued sheets remained unaltered. In the bioadhesive group, histology revealed a smooth fibrin layer in the graft-host interface and a continuous, stratified layer of cytokeratin-3 expressing corneal epithelial cells on the membrane surface. In contrast, suture-fixated membranes showed contracted and prominent membrane edges with epithelial ingrowth into the submembrane interface. CONCLUSION: Our results demonstrate the general feasibility of reproducible and reliable sutureless amniotic membrane fixation onto the corneal surface in rabbits. Stable adherence is maintained until epithelialization is completed. The sutureless technique gives sufficient manipulation time for the sheet before the final cross-linking process is completed. Furthermore, several advantageous characteristics could be demonstrated as increased biocompatibility, better epithelialization pattern and the lack of membrane shrinkage.

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.