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PURPOSE: It has been proposed that changes in the permeability of Bruch's membrane play a role in the pathogenesis of age-related macular degeneration (AMD). This paper investigates, in an in vivo porcine model, the migration of fluorescent latex beads across the Bruch's membrane after subretinal injection.METHODS: Forty-one healthy eyes of 33 three-month-old domestic pigs received a subretinal injection of 0.5, 1.0, 2.0, or 4.0 μm fluorescent latex beads. Between three hours and five weeks after injection evaluations were performed with fundus photographs and histology. Fluorescent beads were identified in unstained histologic sections using the rhodamine filter with the light microscope.RESULTS: The fluorescent latex beads relocated from the subretinal space. Intact beads up to 2.0 μm were found in the choroid, sclera, and extrascleral space. The smaller beads were also found inside choroidal and extrascleral blood vessels. In contrast, the larger beads of 4.0 μm did not pass the Bruch's membrane.CONCLUSION: Subretinally implanted beads up to 2.0 μm pass the Bruch's membrane intact and cross the blood-ocular barrier. The intact beads are found in the choroid, sclera and inside blood vessels. The results give reason to consider the role of subretinal clearance and passage of Bruch's membrane in the development of AMD.

Uveal melanoma (UM) is the most common intraocular malignancy in adults and shows a high rate of metastatic spread. As randomized clinical trials with immune checkpoint inhibitors (ICI) have not been performed in patients with metastatic UM, we analyzed the real-world outcomes in a nationwide population-based study. Clinical data of patients with UM were extracted from the Danish Metastatic Melanoma database, a nationwide database containing unselected records of patients diagnosed with metastatic melanoma in Denmark. Survival before (pre-ICI, n = 32) and after (post-ICI, n = 94) the approval of first-line treatment with ICI was analyzed. A partial response to first-line treatment was observed in 7% of patients treated with anti-programmed cell death protein (PD)-1 monotherapy and in 21% with combined anti-cytotoxic T lymphocyte antigen (CTLA)-4 plus anti-PD-1 therapy. Median progression-free survival was 2.5 months for patients treated in the pre-ICI era compared to 3.5 months in the post-ICI era (hazard ratio (HR) 0.43; 95% confidence interval (CI) 0.28-0.67; p < 0.001). The estimated one-year overall survival rate increased from 25.0% to 41.9% and the median overall survival improved from 7.8 months to 10.0 months, respectively (HR 0.52; 95% CI 0.34-0.79; p = 0.003). Thus, the introduction of ICI as first-line treatment appears to have significantly improved the real-world survival of patients with metastatic UM, despite relatively low response rates compared to cutaneous melanoma. With the lack of therapies proven effective in randomized trials, these data support the current treatment with ICI in patients with metastatic UM.

This study reports findings from a 56-year-old patient, who had received bilateral intravitreal injection of putative adipose tissue-derived stem cells at a private clinic in India with the promise of treatment of NAION. During an observation period of 8, respectively, 18 months, the intravitreally injected cells remained silent in the vitreous bodies without either therapeutic effects or complications. The cells cleared with vitrectomy without evidence of integration in the optic nerve or retina. Contrary to recent reports on patients receiving intravitreal injections of similar putative stem cells with the aim of treating AMD, our patient suffered no devastating ocular consequences. Summary. Contrary to recent reports, this case demonstrated no devastating consequences of intravitreal injection of adipose tissue-derived stem cells during an observation period of up to 18 months. After vitrectomy, the cells cleared without evidence of either harm or integration.

PURPOSE: To determine the size at which choroidal melanomas can metastasize and to report the characteristics of small fatal choroidal melanomas (SFCM).DESIGN: Retrospective case series.METHODS: Ten ocular oncology services submitted 45 patients with a choroidal melanoma 3 mm or less in thickness and 9 mm or less in largest basal diameter (LBD), when treated, who developed metastases.RESULTS: Median tumor thickness was 2.4 mm (range, 1.0-3.0 mm) and LBD 7.3 mm (range, 3.0-9.0 mm). Of 14 (31%) tumors that were first observed, 12 grew a median of 0.5 mm (range, 0.1-1.2 mm) in thickness and 1.0 mm (range, 0-3.0 mm) in LBD within a median of 7 months; 3 were initially smaller than 3 mm in LBD. Number of risk factors for growth and metastasis was 0 for 4% of the tumors; 60% were over 2 mm in thickness, 63% had subretinal fluid, 84% caused symptoms, 57% had orange pigment, and 92% were within 3 mm of the disc. Local recurrence occurred in 8 of 31 eyes (26%) treated conservatively. Median metastasis-free survival was 4.5 years (range, 0.8-15.7 years). Kaplan-Meier estimate of metastasis developing was 15% (95% confidence interval [CI], 7-26), 51% (95% CI, 36-64) and 85% (95% CI, 71-92) by 2, 5, and 10 years, respectively. By the time of analysis, 37 patients had died of metastasis after a median of 7 months.CONCLUSIONS: Choroidal melanomas less than 3.0 mm in LBD are highly unlikely to metastasize. Risk factors of an SFCM are similar to those for all choroidal melanomas of similar size.

Neuropeptide Y (NPY) is a peptide neurotransmitter abundantly expressed in the mammalian retina. Since its discovery, NPY has been studied in retinas of several species, but detailed characterization of morphology, cell-type, and connectivity has never been conducted in larger mammals including humans and pigs. As the pig due to size and cellular composition is a well-suited animal for retinal research, we chose to compare the endogenous NPY system of the human retina to that of pigs to support future research in this field. In the present study, using immunohistochemistry, confocal microscopy and 3D reconstructions, we found NPY to be expressed in GABAergic and calretinin-immunoreactive (-ir) amacrine cells of both species as well as parvalbumin-ir amacrine cells of humans. Furthermore, we identified at least two different types of medium- to wide-field NPY-ir amacrine cells. Finally, we detected likely synaptic appositions between the NPY-ir amacrine cells and melanopsin- and nonmelanopsin-ir ganglion cells, GABAergic and dopaminergic amacrine cells, rod bipolar cells, and horizontal cells, suggesting that NPY-ir cells play diverse roles in modulation of both image and non-image forming retinal signaling. These findings extend existing knowledge on NPY and NPY-expressing cells in the human and porcine retina showing a high degree of comparability. The extensive distribution and connectivity of NPY-ir cells described in the present study further highlights the potential importance of NPY signaling in retinal function.

PURPOSE: To analyze the incidence of adrenal suppression and the glucocorticoid (GC) dose per kilogram body weight given in infants treated with standard protocol for topical ophthalmic GCs after congenital cataract surgery.DESIGN: Retrospective, consecutive case series.PARTICIPANTS: All children younger than 2 years of age who underwent operation for congenital cataract between January 2011 and May 2015 in 1 center.METHODS: Patient charts were reviewed to collect data on results and timing of a standard corticotropin (adrenocorticotropic hormone [ACTH]) stimulation test and GC dose per kilogram body weight.MAIN OUTCOME MEASURES: Incidence of adrenal suppression in children tested on GC treatment. Glucocorticoid dose per kilogram body weight.RESULTS: Among 26 consecutive infants, 15 (58%) were tested while they were still on GC treatment. Ten of these 15 infants (67%) had adrenal suppression, 2 of whom had obvious clinical signs of Cushing's syndrome and 1 of whom had signs of Addisonian crises during general anesthesia. Eleven of the 26 infants (42%) were tested at a median time of 21 days (range, 6-89) after treatment cessation, and they all had normal test results. Children with suppressed adrenal function had received cumulative GC doses per body weight that were significantly higher the last 5 days before testing compared with children with normal test results. Infants with adrenal suppression were treated with hydrocortisone replacement therapy. Adrenal function recovered after a median of 3.1 months (range, 2.3 months to 2.3 years).CONCLUSIONS: Two thirds of the infants tested during treatment with a standard GC protocol after congenital cataract surgery showed adrenal suppression. There was a significant association between the cumulative daily dose of GCs and the test result. Because adrenal suppression is a serious but treatable condition, we recommend a systematic assessment of adrenal function in infants treat

Background: The BRCA1-associated protein-1 (BAP1) tumor predisposition syndrome (BAP1-TPDS) is a hereditary tumor syndrome caused by germline pathogenic variants in BAP1 encoding a tumor suppressor associated with uveal melanoma, mesothelioma, cutaneous melanoma, renal cell carcinoma, and cutaneous BAP1-inactivated melanocytic tumors. However, the full spectrum of tumors associated with the syndrome is yet to be determined. Improved understanding of the BAP1-TPDS is crucial for appropriate clinical management of BAP1 germline variant carriers and their families, including genetic counseling and surveillance for new tumors.Methods: We collated germline variant status, tumor diagnoses, and information on BAP1 immunohistochemistry or loss of somatic heterozygosity on 106 published and 75 unpublished BAP1 germline variant-positive families worldwide to better characterize the genotypes and phenotypes associated with the BAP1-TPDS. Tumor spectrum and ages of onset were compared between missense and null variants. All statistical tests were two-sided.Results: The 181 families carried 140 unique BAP1 germline variants. The collated data confirmed the core tumor spectrum associated with the BAP1-TPDS and showed that some families carrying missense variants can exhibit this phenotype. A variety of noncore BAP1-TPDS -associated tumors were found in families of variant carriers. Median ages of onset of core tumor types were lower in null than missense variant carriers for all tumors combined (P < .001), mesothelioma (P < .001), cutaneous melanoma (P < .001), and nonmelanoma skin cancer (P < .001).Conclusions: This analysis substantially increases the number of pathogenic BAP1 germline variants and refines the phenotype. It highlights the need for a curated registry of germline variant carriers for proper assessment of the clinical phenotype of the BAP1-TPDS and pathogenicity of new variants, thus guiding management of patients and informing

Intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin belong to a heterogenic population of RGCs which regulate the circadian clock, masking behavior, melatonin suppression, the pupillary light reflex, and sleep/wake cycles. The different functions seem to be associated to different subtypes of melanopsin cells. In rodents, subtype classification has associated subtypes to function. In primate and human retina such classification has so far, not been applied. In the present study using antibodies against N- and C-terminal parts of human melanopsin, confocal microscopy and 3D reconstruction of melanopsin immunoreactive (-ir) RGCs, we applied the criteria used in mouse on human melanopsin-ir RGCs. We identified M1, displaced M1, M2, and M4 cells. We found two other subtypes of melanopsin-ir RGCs, which were named "gigantic M1 (GM1)" and "gigantic displaced M1 (GDM1)." Few M3 cells and no M5 subtypes were labeled. Total cell counts from one male and one female retina revealed that the human retina contains 7283 ± 237 melanopsin-ir (0.63-0.75% of the total number of RGCs). The melanopsin subtypes were unevenly distributed. Most significant was the highest density of M4 cells in the nasal retina. We identified input to the melanopsin-ir RGCs from AII amacrine cells and directly from rod bipolar cells via ribbon synapses in the innermost ON layer of the inner plexiform layer (IPL) and from dopaminergic amacrine cells and GABAergic processes in the outermost OFF layer of the IPL. The study characterizes a heterogenic population of human melanopsin-ir RGCs, which most likely are involved in different functions.

PURPOSE: Subretinal perfluorocarbon liquid (PFCL) is a serious complication that can occur after retinal detachment repair. It is possible to remove the PFCL surgically, but retinal damage related to the procedure is unknown. Also, increasing interest in subretinal treatment makes it relevant to examine the functional and morphological consequences of repeated subretinal manipulation. We hypothesized that PFCL in a porcine model can be injected in the subretinal space and removed with minimal effect on retinal structure and function.METHODS: The left eyes of ten healthy three-month-old female domestic pigs were included. Multifocal electroretinograms (mfERG) were recorded before surgery. Following vitrectomy, a PFCL bleb (decalin) was injected subretinally using a 41G cannula. After 14 days the decalin was removed through a 41G cannula in combination with a 2 ml syringe and an intermediate flexible tube. Two weeks after removal, a control mfERG was recorded, the pigs were enucleated and sacrificed and eyes were examined histologically. All statistics were carried out with a paired t-test in SAS Enterprise Guide 7.1® (SAS Institute Inc., Cary, NC, USA).RESULTS: There was no significant difference in mfERG amplitude ratio (left/right eye) between baseline and recordings two weeks after removal of decalin (P1 (M = 0.26, SD = 0.80, p = 0.39), second order kernel (M = -0.18, SD = 0.86, p = 0.57), Direct Response (M = 0.39, SD = 0.61, p = 0.12) or Induced Component (M = -0.03, SD = 0.40, p = 0.80)). Histologically, the photoreceptor outer segments were minimally affected. Otherwise the retina was normal 14 days after removal of decalin. In four pigs the subretinal decalin displaced inferiorly and was no longer accessible for removal.CONCLUSION: Subretinal decalin can be removed within 14 days without lasting retinal damage. Decalin is a heavy liquid where the risk of displacement is high. Future studies using PFCLs to control duration of an experim

Background/aims To present a new method to determine dose depth and the distance from the concave side of the plaque to the tumour base in patients with uveal melanoma treated with ruthenium-106 based on ultrasonic mirror image. Methods We used the mirror image associated with ultrasound during plaque brachytherapy to determine intraobserver reproducibility and interobserver agreement between two surgeons. 230 eyes with primary uveal melanoma were included in a retrospective analysis to determine the distance from the plaque to the tumour base using ultrasound. A phantom study was used to illustrate the effects on radiation dose to apex of the tumour when the dose depth was incorrectly determined. Doses to apex of the tumour were determined using Plaque Simulator. Results The intraobserver variation in dose depth measurement with plaque was significantly lower than for measures without plaque (p<0.001). Agreement between the surgeons was better with a plaque in place. Distances from the plaque to the tumour base were distributed with mean=0.99 (median: 1, range: 0.1-2.9 mm). From the phantom study, it was clear that the tumour did not receive the prescribed 100 Gy if the dose depth was incorrectly determined. Conclusions The dose depth in patients with uveal melanoma must be measured accurately for correct calculation of the radiation dose to the apex of the tumour. Repeated in vivo and in vitro ultrasound measurements of dose depth showed higher variance than measurements using the mirror image produced from a ruthenium plaque. Using the mirror image thus help to improve the dose calculation.

PURPOSE: The time-dependent effect of anesthetics on the retinal function is debated. We hypothesize that in anesthetized animals there is a time-dependent decline that requires optimized multifocal electroretinogram (mfERG) recording procedures.METHODS: Conventional and four-frame global-flash mfERG recordings were obtained approximately 15, 60, and 150 minutes after the induction of propofol anesthesia (20 pigs) and isoflurane anesthesia (nine pigs). In six of the propofol-anesthetized pigs, the mfERG recordings were split in 3-minute segments. Two to 4 weeks after initial recordings, an intraocular injection of tetrodotoxin (TTX) was given and the mfERG was rerecorded as described above. Data were analyzed using mixed models in SAS statistical software.RESULTS: Propofol significantly decreases the conventional and global-flash amplitudes over time. The only significant effect of isoflurane is a decrease in the global-flash amplitudes. At 15 minutes after TTX injection several of the mfERG amplitudes are significantly decreased. There is a linear correlation between the conventional P1 and the global-flash DR mfERG-amplitude (R2 = 0.82, slope = 0.72, P < 0.0001). There is no significant difference between the 3-minute and the prolonged mfERG recordings for conventional amplitudes and the global-flash direct response. The global flash-induced component significantly decreases with prolonged mfERG recordings.CONCLUSIONS: A 3-minute mfERG recording and a single stimulation protocol is sufficient in anesthetized pigs. Recordings should be obtained immediately after the induction of anesthesia. The effect of TTX is significant 15 minutes after injection, but is contaminated by the effect of anesthesia 90 minutes after injection. Therefore, the quality of mfERG recordings can be further improved by determining the necessary time-of-delay from intraocular injection of a drug to full effect.TRANSLATIONAL RELEVANCE: General anesthesia is a

Bilateral diffuse uveal melanocytic proliferation (BDUMP) is a rare paraneoplastic intraocular disease that causes progressive visual loss in patients driven by an IgG factor associated with an underlying malignancy. Characteristic ocular findings include exudative retinal detachment, rapid cataract formation and uveal melanocytic tumours. The awareness and documentation of BDUMP has increased during the past decade, and the increasing amount of data collected demonstrates the effect of treatment with plasmapheresis and the value of diagnostic tools in BDUMP such as genetic and immunologic investigations. The literature of BDUMP has not been reviewed since 2003, and there is a growing need for an updated review on diagnosis and management of BDUMP. We review the literature and report a case of BDUMP with a white ciliary body tumour, iris rubeosis, increased iris pigmentation and cataract.

PURPOSE: This study compared in vivo enhanced-depth imaging optical coherence tomography (EDI-OCT) with ex vivo histology of the choroid in human eyes.METHODS: Three eyes in three patients with advanced iris melanoma without posterior segment involvement underwent EDI-OCT less than 24 hours prior to enucleation and, in one eye, immediately after enucleation. Following fixation in 4% buffered formaldehyde and paraffin embedding, serial sections of the whole eye were cut horizontally, mounted, stained with hematoxylin-eosin and digitized. Alignment between histology and EDI-OCT was made on landmarks such as retinal vessel, the foveal depression, ciliary arteries, drusen, and nevi.RESULTS: Intra- and interindividual variations in relative choroidal thickness were comparable between the two modalities. After histologic fixation of the three melanoma eyes, the fullness of the choroidal vessels was reduced and subfoveal choroid thickness reduced to 56%, 45%, and 56%, respectively, of its in vivo thickness on EDI-OCT.CONCLUSIONS: There were no identifiable discrepancies in choroidal structural patterns between clinical EDI-OCT and histologic sections except that after enucleation and histologic fixation choroidal thickness was reduced to roughly half of its in vivo value, a phenomenon that may reflect the high content of blood vessels in the choroid.

PURPOSE: To collect comprehensive data on choroidal and ciliary body melanoma (CCBM) in children and to validate hypotheses regarding pediatric CCBM: children younger than 18 years, males, and those without ciliary body involvement (CBI) have more favorable survival prognosis than young adults 18 to 24 years of age, females, and those with CBI.DESIGN: Retrospective, multicenter observational study.PARTICIPANTS: Two hundred ninety-nine patients from 24 ocular oncology centers, of whom 114 were children (median age, 15.1 years; range, 2.7-17.9 years) and 185 were young adults.METHODS: Data were entered through a secure website and were reviewed centrally. Survival was analyzed using Kaplan-Meier analysis and Cox proportional hazards regression.MAIN OUTCOME MEASURES: Proportion of females, tumor-node-metastasis (TNM) stage, cell type, and melanoma-related mortality.RESULTS: Cumulative frequency of having CCBM diagnosed increased steadily by 0.8% per year of age between 5 and 10 years of age and, after a 6-year transition period, by 8.8% per year from age 17 years onward. Of children and young adults, 57% and 63% were female, respectively, which exceeded the expected 51% among young adults. Cell type, known for 35% of tumors, and TNM stage (I in 22% and 21%, II in 49% and 52%, III in 30% and 28%, respectively) were comparable for children and young adults. Melanoma-related survival was 97% and 90% at 5 years and 92% and 80% at 10 years for children compared with young adults, respectively (P = 0.013). Males tended to have a more favorable survival than females among children (100% vs. 85% at 10 years; P = 0.058). Increasing TNM stage was associated with poorer survival (stages I, II, and III: 100% vs. 86% vs. 76%, respectively; P = 0.0011). By multivariate analysis, being a young adult (adjusted hazard rate [HR], 2.57), a higher TNM stage (HR, 2.88 and 8.38 for stages II and III, respectively), and female gender (HR, 2.38) independe

Next generation sequencing of uveal melanoma (UM) samples has identified a number of recurrent oncogenic or loss-of-function mutations in key driver genes including: GNAQ, GNA11, EIF1AX, SF3B1 and BAP1. To search for additional driver mutations in this tumor type we carried out whole-genome or whole-exome sequencing of 28 tumors or primary cell lines. These samples have a low mutation burden, with a mean of 10.6 protein changing mutations per sample (range 0 to 53). As expected for these sun-shielded melanomas the mutation spectrum was not consistent with an ultraviolet radiation signature, instead, a BRCA mutation signature predominated. In addition to mutations in the known UM driver genes, we found a recurrent mutation in PLCB4 (c.G1888T, p.D630Y, NM_000933), which was validated using Sanger sequencing. The identical mutation was also found in published UM sequence data (1 of 56 tumors), supporting its role as a novel driver mutation in UM. PLCB4 p.D630Y mutations are mutually exclusive with mutations in GNA11 and GNAQ, consistent with PLCB4 being the canonical downstream target of the former gene products. Taken together these data suggest that the PLCB4 hotspot mutation is similarly a gain-of-function mutation leading to activation of the same signaling pathway, promoting UM tumorigenesis.

Purpose To collect comprehensive data on choroidal and ciliary body melanoma (CCBM) in children and to validate hypotheses regarding pediatric CCBM: children younger than 18 years, males, and those without ciliary body involvement (CBI) have more favorable survival prognosis than young adults 18 to 24 years of age, females, and those with CBI. Design Retrospective, multicenter observational study. Participants Two hundred ninety-nine patients from 24 ocular oncology centers, of whom 114 were children (median age, 15.1 years; range, 2.7-17.9 years) and 185 were young adults. Methods Data were entered through a secure website and were reviewed centrally. Survival was analyzed using Kaplan-Meier analysis and Cox proportional hazards regression. Main Outcome Measures Proportion of females, tumor-node-metastasis (TNM) stage, cell type, and melanoma-related mortality. Results Cumulative frequency of having CCBM diagnosed increased steadily by 0.8% per year of age between 5 and 10 years of age and, after a 6-year transition period, by 8.8% per year from age 17 years onward. Of children and young adults, 57% and 63% were female, respectively, which exceeded the expected 51% among young adults. Cell type, known for 35% of tumors, and TNM stage (I in 22% and 21%, II in 49% and 52%, III in 30% and 28%, respectively) were comparable for children and young adults. Melanoma-related survival was 97% and 90% at 5 years and 92% and 80% at 10 years for children compared with young adults, respectively (P = 0.013). Males tended to have a more favorable survival than females among children (100% vs. 85% at 10 years; P = 0.058). Increasing TNM stage was associated with poorer survival (stages I, II, and III: 100% vs. 86% vs. 76%, respectively; P = 0.0011). By multivariate analysis, being a young adult (adjusted hazard rate [HR], 2.57), a higher TNM stage (HR, 2.88 and 8.38 for stages II and III, respectively), and female gender (HR, 2.38) independently predicted less favorable survival.

TOPIC: This study reviews the evidence behind simulation-based surgical training of ophthalmologists to determine (1) the validity of the reported models and (2) the ability to transfer skills to the operating room.CLINICAL RELEVANCE: Simulation-based training is established widely within ophthalmology, although it often lacks a scientific basis for implementation.METHODS: We conducted a systematic review of trials involving simulation-based training or assessment of ophthalmic surgical skills among health professionals. The search included 5 databases (PubMed, EMBASE, PsycINFO, Cochrane Library, and Web of Science) and was completed on March 1, 2014. Overall, the included trials were divided into animal, cadaver, inanimate, and virtual-reality models. Risk of bias was assessed using the Cochrane Collaboration's tool. Validity evidence was evaluated using a modern validity framework (Messick's).RESULTS: We screened 1368 reports for eligibility and included 118 trials. The most common surgery simulated was cataract surgery. Most validity trials investigated only 1 or 2 of 5 sources of validity (87%). Only 2 trials (48 participants) investigated transfer of skills to the operating room; 4 trials (65 participants) evaluated the effect of simulation-based training on patient-related outcomes. Because of heterogeneity of the studies, it was not possible to conduct a quantitative analysis.CONCLUSIONS: The methodologic rigor of trials investigating simulation-based surgical training in ophthalmology is inadequate. To ensure effective implementation of training models, evidence-based knowledge of validity and efficacy is needed. We provide a useful tool for implementation and evaluation of research in simulation-based training.