Structured illumination ophthalmoscope: super-resolution microscopy on the living human eye.

In this paper, we present the prototype of an ophthalmoscope that uses structured illumination microscopy (SIM) to enable super-resolved imaging of the human retina, and give first insights into clinical application possibilities. The SIM technique was applied to build a prototype that uses the lens of the human eye as an objective to 'super-resolve' the retina of a living human. In our multidisciplinary collaboration, we have adapted this well-established technique in ophthalmology and successfully imaged a human retina using significantly lower light intensity than a state-of-the-art ophthalmoscope. Here, we focus on the technical implementation and highlight future perspectives of this method. A more application-oriented note for physicians on the diagnostic and disease-preventive value of this method, as well as the medical results of the clinical study carried out, will be published in a report addressed to an appropriate specialist audience. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Factors affecting laser power in retinal Navilas laser treatment.

PURPOSE: To evaluate the effect of patient-associated factors on the minimum laser power needed for a mild visible burn in focal laser treatments using the 532 nm Navilas laser system. METHODS: We conducted a monocentric prospective pilot study of 58 eyes of 40 patients with diabetic macular edema. The following parameters were analysed: axial length, refraction, iris pigmentation, lens status, lens grading and densitometry, retinal and choroidal thickness and focus setting during treatment. Laser power was adjusted to produce mild, barely visible burns. Retinal laser burn size was measured 30 min after treatment. RESULTS: Focus setting is significantly correlated with retinal lesion size (r = 0.50, p = 0.001) and laser power (r = 0.44, p < 0.001). Axial length only correlated with laser power when the effect of focus was controlled. Phakic eyes needed more laser power than pseudophakic eyes (78.3 versus 67.2 mW, p = 0.051). No correlation of laser power with any other factor could be found. CONCLUSIONS: Among the examined parameters, focus setting had the strongest effect on the laser power needed to produce a mild visible burn. The association of focus with laser power can be explained by the focus-dependent change of retinal spot size. Lens status (phakic versus pseudophakic patients) seems to influence laser light transmission in the examined age group.

Subfoveal choroidal tuberculoma in a healthy young woman caused by Mycobacterium bovis.

PURPOSE: To describe a case of a macular tuberculoma in a young and healthy woman after developing a tuberculous lymphadenitis caused by Mycobacterium bovis. METHODS: Retrospective case report. RESULTS: Tuberculous lymphadenitis caused by Mycobacterium bovis was detected after biopsy and histological examination as well as PCR testing of cervical lymph nodes in a 20 year-old patient. An interferon gamma was positive. Shortly starting anti-tubercular therapy (ATT), the patient developed visual deterioration caused by a single yellowish subretinal structure in the macula of the right eye. Optical coherence tomography showed a dome-shaped, hyperreflective, subfoveal choroidal lesion with subtle subretinal fluid. ATT with isoniazid, rifampicin, ethambutol, and pyrazinamide was performed, and the de-escalation therapy with isoniazid and rifampicin was extended to 7 months. Further examinations showed regression of choroidal tuberculoma to a scar. CONCLUSION: This is the first reported case of choroidal tuberculoma after tuberculous lymphadenitis caused by Mycobacterium bovis.

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.

Light-emitting diode technology in vitreoretinal surgery.

BACKGROUND: Systems for vitreoretinal illumination during surgery usually consist of an external light source and a light fiber. We introduce a new illumination system for vitreoretinal surgery based on the light-emitting diode technology, with an embedded light source in the handle of the light fiber, making a separate light source unnecessary. METHODS: A prototype of a new illumination system for vitreoretinal surgery (ocuLED; Geuder, Heidelberg, Germany) was tested. This system consists of a handle with a built-in light-emitting diode, supported by an external power source. The OcuLED was analyzed in regards to wavelength, maximum radiant power, and maximum irradiance and was compared with three commercially available vitreoretinal illumination systems. Furthermore, the first intraoperative application and handling were evaluated. RESULTS: The ocuLED system works with a cool white or a neutral white light-emitting diode and is powered externally. The wavelength spectrum shows a maximum at 565 nm and a second peak at 455 nm. Compared with other light sources, the proportion of potentially harmful blue light is low. Maximum radiant power and irradiance are in line with xenon and mercury vapor light sources. The intrasurgical light is bright and offers good visibility. The handle of ocuLED is slightly wider than commonly used light fiber handles, which do not affect its use during surgery. CONCLUSION: Technical progress in light-emitting diode technology allows minimizing the equipment for vitreoretinal illumination. The OcuLED provides bright illumination without an external light source. Wavelength spectrum, maximum radiant power, and irradiance are safe from the risk of phototoxic damage. Intrasurgical handling is identical to conventional light fibers.

OCT patterns of macular edema and response to bevacizumab therapy in retinal vein occlusion.

BACKGROUND: Baseline OCT morphology of macular edema (ME) due to branch (BRVO) or central retinal vein occlusion (CRVO) was evaluated with respect to response to bevacizumab treatment. METHODS: Sixty-five patients (33 CRVO, 32 BRVO) were treated with intravitreal injections of 2.5 mg bevacizumab. Reinjections were only performed if ME persisted or recurred. Follow-up ranged from 23 to 128 weeks. OCT (Stratus OCT™, Carl Zeiss Meditec) morphology at baseline was analyzed retrospectively to evaluate its effect on treatment outcome (ETDRS visual acuity and central retinal thickness). Baseline OCT scans were studied with regard to central retinal thickness (CRT), presence and height of subretinal fluid (SRF), intraretinal cystoid spaces (ICS) and maximum horizontal diameter of the largest delimitable ICS. RESULTS: In CRVO patients, baseline CRT is not correlated with CRT at last visit, but there is a significant correlation of baseline CRT with final visual acuity (VA). Presence of SRF and diameter of ICS do not influence functional and morphological response to bevacizumab treatment. Baseline CRT in BRVO patients is not significantly correlated with final VA and final CRT. There is no difference in treatment response between patients with or without baseline SRF. BRVO patients with large ICS >600 µm (13.8%) at baseline had a significantly worse VA at last visit than patients with smaller ICS (p = 0.011), and did not achieve a significant improvement under bevacizumab therapy. The duration of retinal vein occlusion before start of treatment was significantly longer in patients with ICS >600 µm (232 ± 96 weeks versus 17 ± 17 weeks; p < 0.001). There is no correlation of ICS diameter and duration of vein occlusion in patients with ICS <600 µm. CONCLUSIONS: In CRVO patients, baseline CRT and final VA are significantly correlated, whereas the presence of SRF or diameter of ICS was not predictive for treatment outcome. In BRVO patients, ICS with a diameter of >600 µm are associated with a long duration of vein occlusion and poor functional response to treatment with bevacizumab. CRT and SRF were not found to be significant prognostic factors.

Predictive factors for changes in macular edema in intravitreal bevacizumab therapy of retinal vein occlusion.

BACKGROUND: To evaluate prognostic factors of response to intravitreal bevacizumab therapy of macular edema (ME) due to central retinal vein occlusion (CRVO) or branch retinal vein occlusion (BRVO). METHODS: Patients with ME due to CRVO (32 patients) or BRVO (38 patients) received intravitreal bevacizumab (2.5 mg/0.1 ml) at baseline, and every 6 to 8 weeks if OCT showed persistent or recurrent ME. Visual acuity (EDTRS), ophthalmic examination and OCT were performed at baseline and at all follow-up visits. Six to 8 weeks after first injection, baseline factors (visual acuity, central retinal thickness, age and gender) were analyzed retrospectively between patients with resolved ME (group 1) and persisting ME (group 2). At last visit, baseline factors of patients with resolved ME since first injection (group A), with recurrent ME since baseline (group B) and with persistent ME since baseline (group C) were compared. RESULTS: In CRVO patients, central retinal thickness (CRT) and patients' age are prognostic predictors in bevacizumab therapy. Age of CRVO patients differed significantly between groups 1 and 2 after first injection, while CRT only showed a strong trend to thinner CRT. At last visit, age and CRT differed statistically significantly between groups A, B and C. In BRVO patients, none of the investigated factors revealed any prognostic value. In CRVO and BRVO patients, final CRT is correlated with the CRT after first injection. CONCLUSION: CRT and age of patients have prognostic value in bevacizumab therapy of ME due to CRVO. CRVO patients who benefit from therapy are significantly younger and have a lower CRT at baseline than patients with persisting ME. In BRVO patients, no predictive factors for effectiveness of bevacizumab therapy could be observed.

Intravitreal bevacizumab in vascular pigment epithelium detachment as a result of subfoveal occult choroidal neovascularization in age-related macular degeneration.

PURPOSE: The purpose of this study was to evaluate the effect of intravitreally administered bevacizumab on untreated vascularized pigment epithelium detachment (PED) in sub- or juxtafoveal occult choroidal neovascularization as a result of age-related macular degeneration. METHODS: In this retrospective study, 28 untreated eyes of 26 patients (4 men, 22 women; mean age, 74.6 ± 7.2 years) with PED and sub- or juxtafoveal occult choroidal neovascularization as a result of age-related macular degeneration and additional intra- and/or subretinal fluid were treated with intravitreal injections of 1.25 mg bevacizumab. Baseline and follow-up visits included best-corrected visual acuity, complete ophthalmic examination, and Stratus optical coherence tomography. Fluorescein angiography was performed at baseline. Reinjections were performed if intra- and/or subretinal fluid persisted or recurred or PED increased. RESULTS: Patients received 3.2 ± 1.8 injections (follow-up 37.9 ± 18.3 weeks). Mean maximum PED height showed a tendency to decrease (372 ± 150.5 µm to 290.3 ± 189 µm; P = 0.094). In 14 eyes (53.8%), PED height was reduced at last visit, including complete flattening in 1 eye. Mean visual acuity remained stable (0.58 ± 0.30 logarithm of the minimum angle of resolution to 0.58 ± 0.37 logarithm of the minimum angle of resolution; P = 0.905). Pigment epithelium detachment response to treatment did not correlate with baseline PED height or visual acuity at baseline or at the last visit. One patient sustained a retinal pigment epithelium rip, and another patient sustained an extensive subretinal hemorrhage. CONCLUSION: During bevacizumab therapy, mean PED height decreases in 50% of patients. No predictive factors for the response of PED to bevacizumab treatment could be identified. Treatment of PED with bevacizumab might result in a long-term functional benefit compared with the natural course.

Long-term follow-up of OCT-guided bevacizumab treatment of macular edema due to retinal vein occlusion.

BACKGROUND: To evaluate the long-term outcome of an OCT-guided reinjection scheme for bevacizumab treatment of macular edema (ME) due to retinal vein occlusion. METHODS: Patients with persistent ME (>250 microm) due to central retinal vein occlusion (CRVO) or branch retinal vein occlusion (BRVO) received intravitreal bevacizumab 2.5 mg/0.1 ml. Visual acuity (ETDRS), ophthalmic examination and OCT were performed at baseline and at 6- to 8-week intervals. Reinjections were only performed if OCT showed persistent or recurrent ME. RESULTS: Sixty-one patients with a minimum follow-up of 25 weeks were included in this analysis. Mean follow-up was 60 +/- 29 wks. In CRVO patients, central retinal thickness (CRT) decreased from 748 +/- 265 microm to 372 +/- 224 microm (p < 0.001) and visual acuity (VA) improved by 1.9 +/- 3.2 lines. In BRVO patients, mean CRT decreased from 601 +/- 206 microm to 386 +/- 178 microm (p < 0.001) and VA improved by 1.8 +/- 2.6 lines. Thirty-three percent of CRVO and 15% of BRVO patients did not show a ME recurrence for > or =25 wks at last visit. Thirty-seven percent of CRVO and 50% of BRVO patients suffered recurrences of ME within the last 25 wks, whereas 30% of CRVO and 35% of BRVO patients did not achieve a complete resolution of ME at any follow-up visit after receiving a minimum of three injections. CRVO patients with dry interval of > or =25 weeks at last visit were significantly younger, had a thinner CRT at baseline and more often had a complete resolution of ME after the first injection. In CRVO and BRVO, final VA was correlated significantly with initial VA, patients' age and final CRT. Change of VA was correlated with change of CRT in BRVO. CONCLUSIONS: Patients with retinal vein occlusion benefit from treatment with bevacizumab. Favourable long-term results without necessity of further injections were achieved in 33% and 15% of CRVO and BRVO patients respectively. The remaining patients needed repeated injections to treat ME recurrences. However, one third of the CRVO/BRVO patients did not improve in VA, and further injections might be discontinued in these patients.

Intravitreal bevacizumab for treatment of chronic central serous chorioretinopathy.

PURPOSE: To evaluate the short-term safety and efficacy of intravitreal bevacizumab for the treatment of intraretinal or subretinal fluid accumulation secondary to chronic central serous chorioretinopathy (CSC). METHODS: Twelve patients were treated with intravitreal injections of 2.5 mg bevacizumab at 6- to 8-week intervals until intraretinal or subretinal fluid resolved. Observation procedures were Early Treatment Diabetic Retinopathy Study best-corrected visual acuity (BCVA), ophthalmic examination, and optical coherence tomography (OCT), performed at 6- to 8-week intervals. Fluorescein angiography was performed at baseline visit and thereafter depending on clinical and OCT findings. Multivariate analysis of variance with repeated measures was used to calculate a statistical significance of change in BCVA and mean central retinal thickness, which were the main outcome measures. SAS statistical software was used for analyses. RESULTS: Patients received 2-/+1 intravitreal injections of bevacizumab on average during a follow-up of 24-/+14 weeks. Mean BCVA increased by 2-/+2 lines; the change in BCVA (logMAR) was significant (p<0.02). Mean central retinal thickness decreased significantly over follow-up (p<0.05), with 6 patients (50%) showing complete resolution of subretinal fluid. CONCLUSIONS: Anatomic and functional improvement following intravitreal bevacizumab injections suggest that vascular endothelial growth factor (VEGF) may be involved in fluid leakage in patients with chronic CSC. The results suggest a possible role for anti-VEGF agents in the treatment of chronic CSC. Further evaluation of intravitreal bevacizumab for chronic CSC in controlled randomized studies is warranted.

Palliative radiotherapy of retrobulbar orbit metastases due to breast cancer.

BACKGROUND: In breast cancer, the occurrence of retrobulbar metastases of the orbit is rare compared to intraocular metastases. The clinical symptoms are quite different. Impairment of vision, exophthalmus, double vision, vertigo, and pain reduce patients' quality of life. PATIENTS AND METHODS: The benefit of palliative irradiation of the orbit was researched retrospectively in 7 patients. This report also presents the first case in the literature of a breast cancer patient with bi-orbital enophthalmus caused by bilateral retrobulbar metastases that were successfully treated with radiotherapy. Irradiation was performed by photon or electron beams (20-50 Gy). Clinical restaging was done at the end of radiotherapy and 6 weeks thereafter. RESULTS: After irradiation, 6 out of 7 patients showed a distinct clinical response with good palliation and no major side effects. Exophthalmus, pain, and vertigo were significantly reduced in all cases. Double vision disappeared in 3 out of 4 patients, eye muscle paralysis in 5 out of 6 patients. The median overall survival after irradiation of the orbit was 7.3 months. CONCLUSION: Palliative radiotherapy of retrobulbar metastases of breast cancer is very effective in reducing acute clinical symptoms and increasing quality of life. Nonetheless, patients have a poor prognosis.

Outcomes of surgical interventions for primary childhood glaucoma in Northern Tanzania.

AIM: To evaluate goniotomy, trabeculotomy, transscleral cyclophotocoagulation (TSCPC) and trabeculectomy as initial procedures in children with primary childhood glaucoma from Northern Tanzania. METHODS: A retrospective analysis of consecutive interventions for primary childhood glaucoma from 2000 to 2013 was conducted at the Kilimanjaro Christian Medical Centre. Success was defined as postoperative intraocular pressure (IOP) below 22 mmHg. Success rates, IOP, visual acuity (VA), subsequent interventions and potential risk factors for failure were reported for the respective interventions. RESULTS: The study included 116 eyes of 70 children (age 4.6±5.9 years) with primary childhood glaucoma; 46 (65.7%) children were male. The preoperative IOP was 33.1±10.2 mmHg, the preoperative cup/disc (CD) ratio 0.71±0.3 and the corneal diameter 13.3±1.4 mm. As a primary intervention, 61 (52.6%) eyes underwent goniotomy, 10 (8.6%) eyes trabeculotomy, 12 (10.3%) TSCPC and 33 (28.4%) trabeculectomy. Follow-up data after 12 months were available for 63 (54.3%) eyes. Success rates at 12 months were 38% (goniotomy), 30% (trabeculotomy), 17% (TSCPC) and 64% (trabeculectomy). All interventions achieved a statistically significant IOP reduction at 3, 6 and 12 months, except for trabeculotomy after 6 months and TSCPC at all time points. Postoperative endophthalmitis occurred in one child treated with trabeculectomy. VA in 82% of all eyes was maintained or had improved after 12 months. CONCLUSIONS: Based on the success rates of this retrospective analysis, goniotomy or trabeculotomy for younger and trabeculectomy for older children can be recommended in our setting to reduce IOP. Late presentation in combination with advanced glaucomatous damage as well as erratic postoperative follow-up and treatment were likely factors that compromised overall success rates. More efforts are necessary to detect the blinding disease earlier and improve adherence to follow-up.

Trabeculectomy or Transscleral Cyclophotocoagulation as Initial Treatment of Secondary Childhood Glaucoma in Northern Tanzania.

PURPOSE: The purpose is to describe the outcome of trabeculectomy with transscleral cyclophotocoagulation (TSCPC) as an initial intervention for secondary childhood glaucoma in Northern Tanzania. METHODS: A retrospective, consecutive case series was analyzed of all children with secondary childhood glaucoma who underwent initial trabeculectomy or TSCPC between 2000 and 2013 at a referral eye unit in Northern Tanzania. Retrospective data were collected on causes of glaucoma, intraocular pressure (IOP), visual acuity, complications, and subsequent interventions. Outcomes were evaluated using Kaplan-Meier survival analysis and compared with Cox regression analysis. The main outcome measure was failure (IOP>21 mm Hg). RESULTS: Thirty-six eyes of 27 children (male, 21; median age, 9 y; range, 0.3 to 15 y) with secondary childhood glaucoma underwent trabeculectomy (19 eyes, 53%) or TSCPC (17 eyes, 47%). Causes included ocular trauma (13, 36%), previous cataract surgery (12, 33%), congenital aniridia (5, 14%), Sturge-Weber syndrome (2, 6%), steroid-induced glaucoma (2, 6%), uveitis (1, 3%), and unspecified leucoma (1, 3%). After 12 months, success was achieved in 48% after trabeculectomy and 18% after TSCPC, with visual acuity remaining unchanged in 11 of 14 (79%) and 4 of 5 eyes (80%), respectively. One third of the children did not return for follow-up after 1 year. Distance to the hospital (>100 km) was a significant risk factor for trabeculectomy failure (P=0.031). CONCLUSIONS: A high proportion of secondary childhood glaucoma in Northern Tanzania was caused by trauma and previous cataract surgery. Trabeculectomy was associated with better IOP control but also a higher complication rate. The ability to maintain visual function was comparable after both interventions. Failure was associated with a journey to the eye hospital (>100 km) possibly leading to late presentation with advanced disease and erratic follow-up.

Successful hormone treatment of orbital leiomyoma.

PURPOSE: To describe the successful hormone treatment of noncircumscribed orbital leiomyoma. DESIGN: Case report. PARTICIPANT: A 66-year-old woman with histologically proven orbital leiomyoma. METHODS: Because surgical excision of the tumor was impossible, therapy with the gonadotropin-releasing hormone (GnRH) analog goserelin was performed. RESULTS: Hormone treatment was well tolerated and no side effects were noted. The tumor regressed significantly during treatment. No recurrence occurred during a 5-year follow-up period. CONCLUSIONS: This report describes for the first time successful hormone treatment of a noncircumscribed orbital leiomyoma. Goserelin may have an antiproliferative effect on leiomyoma cells via membrane receptors for GnRH.

Intraocular Pharmacokinetics of Aflibercept and Vascular Endothelial Growth Factor-A.

PURPOSE: To determine intraocular pharmacokinetics of aflibercept and VEGF-A in patients with neovascular age-related macular degeneration (nAMD) during a treatment period of 6 months. METHODS: Seven nonvitrectomized patients diagnosed with macular edema secondary to nAMD undergoing intravitreal injections (IVI) of aflibercept. Patients were treatment naïve at least for the last 2 months and received intravitreal injection of 2 mg aflibercept for the first time. Aqueous humor samples were obtained prior to each injection procedure during a 6-month period: three times monthly, then bimonthly. Over all 35 samples were analyzed with ELISA for unbound VEGF-A and a self-developed assay for unbound aflibercept. RESULTS: In all cases, wet AMD was inactive after IVI. Unbound aflibercept could be detected in all samples. Initial mean concentration of aflibercept was 305.4 ± 43.8 µg/mL and remained stable after the first injection with 0.8 ± 0.5 µg/mL. Initial mean level of unbound VEGF-A was 190.7 ± 26.9 pg/mL. A significant decrease of the concentration to 92.6 ± 10.2 pg/mL (P < 0.05, Wilcoxon rank sum test) after the first injection was observed. This level remained stable during further treatment. CONCLUSIONS: Levels of unbound aflibercept and unbound VEGF-A remained stable after every month and every second month of IVI. The findings of these small case series support suggestions that treatment intervals with bimonthly IVI of aflibercept are sufficient due to a detectable remaining biologic active concentration of aflibercept.

Interferon alpha 2b decreases hepatic micrometastasis in a murine model of ocular melanoma by activation of intrinsic hepatic natural killer cells.

PURPOSE: To investigate in a murine model the mechanism by which micrometastatic melanoma, which spreads from the eye to the liver, is controlled by interferon (IFN)-alpha 2b. METHODS: Major histocompatibility (MHC) class I antigen (H-2, all haplotypes) expression in three murine melanoma cell lines (Queens, B16LS9, B16F10) was determined by flow cytometric immunophenotyping. The cell lines were heterotopically inoculated into the posterior compartments (PCs) of C57Bl/6 mice, and the mice were given intraperitoneal (IP) injections of IFN-alpha 2b or PBS for 1 or 4 days before enucleation at 7 days after inoculation. Groups of mice were made NK deficient or depleted with subcutaneous (s.c.) injection of anti-asialo GM1. The mice were killed at 28 days or 56 days (survival experiment) after inoculation, and the number of hepatic micrometastases was histologically determined. NK cells were isolated from the spleen and liver at necropsy, and propidium iodide labeled target-specific cytolysis was determined by flow cytometry. The micrometastases were evaluated for apoptosis and proliferation with TUNEL and MIB1 immunostaining, respectively, and TUNEL-to-MIB1 ratios were determined. Hepatic NK cells were immunostained with CD49b. RESULTS: MHC class I antigen was expressed in the three cell lines in the order of Queens < B16LS9 < B16F10. All cell lines grew, were confined to the PC, and formed hepatic micrometastases. A decrease in micrometastases, an increase in target-specific cytolysis, and an increase in survival correlated with decreased HLA class I expression by the melanoma cells. The IFN-alpha 2b treatment resulted in a boost of intrinsic hepatic NK cells, demonstrated in NK-deficient but not NK-depleted mice. The treatment effect corresponded to increased apoptosis (TUNEL)-proliferation (MIB1) ratios in the micrometastases. Immunostaining demonstrated an increased number of intrahepatic NK cells associated with the micrometastases in treated groups. CONCLUSIONS: Neoadjuvant IFN-alpha 2b results in decreased hepatic micrometastasis and increased survival time through increased intrinsic hepatic NK cell-mediated tumor apoptosis in a murine model of metastatic ocular melanoma.

Low dose adjuvant angiostatin decreases hepatic micrometastasis in murine ocular melanoma model.

PURPOSE: To investigate the effect of different doses of adjuvant angiostatin affecting hepatic micrometastasis in a murine model of metastatic ocular melanoma. METHODS: Angiostatin and plasminogen expression was detected in three murine melanoma cell lines (Queens, B16F10, and B16LS9). The three cell lines were heterotopically inoculated into the posterior compartment (PC) of the right eyes of C57BL/6 mice. After enucleation, the mice were given injections of 100 microl PBS and low dose (0.1 microg/microl) or high dose (0.3 microg/microl) murine recombinant angiostatin every day for 14 days after enucleation. The mice were sacrificed at 21 days post-enucleation and hepatic micrometastases were counted. In vitro migration/invasion assays were performed with low (0.1 microg/microl) and high (50 microg/microl) concentration angiostatin supplementation. Quantitative RT-PCR detected mRNA and Western analysis determined protein expression of VEGF for all cell lines. Evaluation of TdT mediated dUTP nick end labeling (TUNEL) and MIB1 immunostaining of the micrometastases determined apoptosis and proliferation ratios. RESULTS: There was a decrease in micrometastasis in the low dose group for Queens (p<0.05), B16F10 (p<0.05), and B16LS9 melanoma (p<0.01) cell lines. Two of the cell lines (B16F10 and B16LS9) elaborated plasminogen and were able to cleave plasminogen into K1-K4 (angiostatin). There was a decrease in the in vitro migration and invasion after supplementation with low concentration compared to high concentration angiostatin (p<0.01). VEGF mRNA and protein expression decreased in all cells lines in low concentration angiostatin, with the greatest decrease in B16LS9 cells (p<0.05). Apoptosis ratios were increased (p<0.01) and proliferation ratios were decreased (p<0.01) in hepatic micrometastases after treatment with low dose angiostatin. CONCLUSIONS: There were significantly fewer micrometastases in treated compared to controls with low dose compared to high dose angiostatin. This treatment results in apoptosis in the micrometastases. The mechanism appears to be related an anti-migratory effect and altered VEGF expression by melanoma cells.

Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization.

PURPOSE: To determine the expression of angiogenic cytokines in macrophages and retinal pigment epithelium cells in choroidal neovascularization (CNV). METHODS: Ten surgically-excised subfoveal CNV specimens and ten eye bank eyes with subfoveal CNV were routinely processed, serially sectioned, and immunostained for factor VIII (F8), CD68 (KP1), cytokeratin 18 (CK18), vascular endothelial growth factor (VEGF), tissue factor (TF), and monocyte chemotactic protein (MCP). The CNV was classified as "inflammatory active" (more inflammation than fibrosis) or "inflammatory inactive" (morefibrosis than inflammation). The immunostaining was graded as none, mild (+), moderate (++), or heavy (+++). Five additional surgically-excised CNV specimens were dual labeled with CK18/MCP or CD68/TF and confocal scanning laser microscopy was performed. RESULTS: Vascular endothelium, macrophages, and RPE expressed F8, KP1, and CK18 respectively. Macrophages expressed + to ++ VEGF and ++ to +++ TF; RPE expressed ++ to +++ VEGF and ++ to +++ MCP. Staining for angiogenic cytokines was stronger in inflammatory active versus inflammatory inactive CNV. RPE dual labeled for CK18/MCP and macrophages dual labeled for CD68/TF. CONCLUSIONS: This study shows that RPE cells express MCP, a cytokine involved with macrophage recruitment, and that macrophages express TF in CNV. Macrophages and RPE express VEGF, thus perpetuating angiogenesis. TF is involved with fibrin formation and provides a scaffold effect for growth of the CNV complex. CNV likely represents a dynamic process with inflammatory active and inflammatory inactive (involutional) stages.

High-resolution imaging of autofluorescent particles within drusen using structured illumination microscopy.

PURPOSE: Autofluorescent (AF) material within drusen has rarely been described and there is little knowledge about origin and formation of these particles. Drusen formation is still a relatively unknown process and analysis of AF inclusions might be important for the understanding of fundamental processes. Here we present a detailed analysis of drusen containing AF material using structured illumination microscopy (SIM), which provides a lateral resolution twice as high as conventional fluorescence microscopy. METHODS: Eight histological retinal pigment epithelium (RPE) sections obtained from eight human donor eyes (76 ± 4 years) were examined by SIM using laser light of different wavelengths (488 nm, 568 nm). Drusen were studied with regards to their size and shape. AF material within drusen was analysed in terms of size, shape, AF behaviour, and distribution across drusen. RESULTS: A total of 441 drusen were found, of which 101 contained AF material (22.9%). 90.1% of these drusen were smaller than 63 µm (mean: 35.65 µm ± 2.38 µm) regardless of whether classified as hard or soft drusen. AF particles (n=190) within drusen show similar spectra compared with lipofuscin granules in RPE cells. Up to 11 particles were found within a single druse. Nearly all particles were located in the outer 2/3 of the drusen (85.94%). CONCLUSIONS: SIM allows studying AF particles within drusen on a higher resolution level compared with conventional fluorescence, multiphoton or even confocal microscopy and therefore provides detailed insights in drusen. Shape and autofluorescence analysis of the material embedded in drusen suggest that these particles originate from the overlaying RPE cells.