Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression

The objective of the present study was to develop a quantitative method to evaluate laser-induced choroidal neovascularization (CNV) in a rat model using Heidelberg Retina Angiograph 2 (HRA2) imaging. The expression of two heparan sulfate proteoglycans (HSPG) related to inflammation and angiogenesis was also investigated. CNV lesions were induced with argon laser in 21 heterozygous Zucker rats and after three weeks a fluorescein angiogram and autofluorescence exams were performed using HRA2. The area and greatest linear dimension were measured by two observers not aware of the protocol. Bland-Altman plots showed agreement between the observers, suggesting that the technique was reproducible. After fluorescein angiogram, HSPG (perlecan and syndecan-4) were analyzed by real-time RT-PCR and immunohistochemistry. There was a significant increase in the expression of perlecan and syndecan-4 (P < 0.0001) in retinas bearing CNV lesions compared to control retinas. The expression of these two HSPG increased with increasing CNV area. Immunohistochemistry demonstrated that the rat retina damaged with laser shots presented increased expression of perlecan and syndecan-4. Moreover, we observed that the overexpression occurred in the outer layer of the retina, which is related to choroidal damage. It was possible to develop a standardized quantitative method to evaluate CNV in a rat model using HRA2. In addition, we presented data indicating that the expression of HSPG parallels the area of CNV lesion. The understanding of these events offers opportunities for studies of new therapeutic interventions targeting these HSPG.

A heparin mimetic isolated from a marine shrimp suppresses neovascularization.

BACKGROUND: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age-related macular degeneration (AMD). Heparin/heparan sulfate are known to play important roles in neovascularization due to their abilities to bind and modulate angiogenic growth factors and cytokines. Previously, we have isolated from marine shrimp a heparin-like compound with striking anti-inflammatory action and negligible anticoagulant and hemorrhagic activities. OBJECTIVES: To investigate the role of this novel heparin-like compound in angiogenic processes. METHODS AND RESULTS: The anti-angiogenic effect of this heparinoid in laser-induced CNV and in vitro models is reported. The compound binds to growth factors (FGF-2, EGF and VEGF), blocks endothelial cell proliferation and shows no cytotoxic effect. The decrease in proliferation is not related to cell death either by apoptosis or secondary necrosis. The results also showed that the heparinoid modified the 2-D network organization in capillary-like structures of endothelial cells in Matrigel and reduced the CNV area. The effect on CNV area correlates with decreases in the levels of VEGF and TGF-ß1 in the choroidal tissue. The low content of 2-O-sulfate groups in this heparinoid may explain its potent anti-angiogenic effect. CONCLUSIONS: The properties of the shrimp heparinoid, such as potent anti-angiogenic and anti-inflammatory activities but insignificant anticoagulant or hemorrhagic actions, point to this compound as a compelling drug candidate for treating neovascular AMD and other angioproliferative diseases. A mechanism for the anti-angiogenic effect of the heparinoid is proposed.

Quantitative evaluation of experimental choroidal neovascularization by confocal scanning laser ophthalmoscopy: fluorescein angiogram parallels heparan sulfate proteoglycan expression.

The objective of the present study was to develop a quantitative method to evaluate laser-induced choroidal neovascularization (CNV) in a rat model using Heidelberg Retina Angiograph 2 (HRA2) imaging. The expression of two heparan sulfate proteoglycans (HSPG) related to inflammation and angiogenesis was also investigated. CNV lesions were induced with argon laser in 21 heterozygous Zucker rats and after three weeks a fluorescein angiogram and autofluorescence exams were performed using HRA2. The area and greatest linear dimension were measured by two observers not aware of the protocol. Bland-Altman plots showed agreement between the observers, suggesting that the technique was reproducible. After fluorescein angiogram, HSPG (perlecan and syndecan-4) were analyzed by real-time RT-PCR and immunohistochemistry. There was a significant increase in the expression of perlecan and syndecan-4 (P < 0.0001) in retinas bearing CNV lesions compared to control retinas. The expression of these two HSPG increased with increasing CNV area. Immunohistochemistry demonstrated that the rat retina damaged with laser shots presented increased expression of perlecan and syndecan-4. Moreover, we observed that the overexpression occurred in the outer layer of the retina, which is related to choroidal damage. It was possible to develop a standardized quantitative method to evaluate CNV in a rat model using HRA2. In addition, we presented data indicating that the expression of HSPG parallels the area of CNV lesion. The understanding of these events offers opportunities for studies of new therapeutic interventions targeting these HSPG.

Twelve-month short-term safety and visual-acuity results from a multicentre prospective study of epiretinal strontium-90 brachytherapy with bevacizumab for the treatment of subfoveal choroidal neovascularisation secondary to age-related macular degeneration.

BACKGROUND/AIMS: This study evaluated the short-term safety and feasibility of epiretinal strontium-90 brachytherapy delivered concomitantly with intravitreal bevacizumab for the treatment of subfoveal CNV due to AMD for 12 months. A 3-year follow-up is planned. METHODS: In this prospective, non-randomised, multicentre study, 34 treatment-naïve patients with predominantly classic, minimally classic and occult subfoveal CNV lesions received a single treatment with 24 Gy beta radiation (strontium-90) and two injections of the anti-VEGF antibody bevacizumab. Adverse events were observed. BCVA was measured using standard ETDRS vision charts. RESULTS: Twelve months after treatment, no radiation-associated adverse events were observed. In the intent-to-treat (ITT) population, 91% of patients lost <3 lines (15 ETDRS letters) of vision at 12 months, 68% improved or maintained their BCVA at 12 months, and 38% gained >/=3 lines. The mean change in BCVA observed at month 12 was a gain of 8.9 letters. CONCLUSION: The safety and efficacy of intraocular, epiretinal brachytherapy delivered concomitantly with anti-VEGF therapy for the treatment of subfoveal CNV secondary to AMD were promising in this small study population. Long-term safety will be assessed for 3 years. This regimen is being evaluated in a large, multicentre, phase III study.

Experimental selective choriocapillaris photothrombosis using a modified indocyanine green formulation.

BACKGROUND: This in vivo study assessed and compared the effectiveness of an aqueous indocyanine green (ICG) formulation (R-ICG) and a lipid ICG formulation (L-ICG) in occluding the rabbit choriocapillaris, and determined the singlet oxygen quantum yields and aggregation properties of both formulations in vitro. METHODS: Singlet oxygen production and aggregation were compared. The eye fundus of 30 albino rabbits was irradiated 0-15 min after dye injection using an 810 nm diode laser. Fluorescein angiography and light microscopy were used to evaluate the safety and efficacy of R-ICG and L-ICG. RESULTS: L-ICG decreased the dimerisation constant and the tendency of ICG to form aggregates, and increased the efficiency of ICG in generating singlet oxygen (R-ICG, PhiDelta = 0.120 and L-ICG, PhiDelta = 0.210). Using a 10 mg/kg dose, choriocapillaris occlusion was achieved at a light dose of 35.8 J/cm(2) with L-ICG and 71.6 J/cm(2) with R-ICG with minimal damage to the neurosensory retina. CONCLUSION: Restrictions to the use of ICG in aqueous solution, low singlet oxygen quantum yields and high aggregation tendency, were overcome with L-ICG. The lower laser irradiance required to obtain choriocapillaris occlusion may suggest that L-ICG is a more potent and selective photosensitiser than R-ICG.

Optical coherence tomography characteristics of full-thickness traumatic macular holes.

PURPOSE: The objective of this paper is to describe the optical coherence tomography (OCT) characteristics of patients with full-thickness traumatic macular hole (TMH) and to correlate them with biomicroscopy findings. METHODS: Twelve eyes of ten consecutive patients with full-thickness TMH participated in this observational retrospective multicentre study. Patients underwent biomicroscopic fundus examination, colour fundus photography, and OCT. RESULTS: Traumatic macular hole was documented with OCT in five women and five men. Mean (range) time between trauma and macular hole (MH) diagnosis was 8.1 (1-24) months. The shape of TMHs was round in 11 (91.7%) eyes. The posterior vitreous was completely detached in six (50%) eyes, and with an operculum in one (8.3%) eye. The common findings seen on OCT were: (1) full-thickness loss of retinal tissue through the hole with sharp edges, perpendicular to the retinal pigment epithelium in five (41.7%) eyes; (2) TMH with an operculum totally detached from the hole's edge in two (16.7%) eyes; (3) presence of epiretinal membrane around of the hole in three (25%) eyes; and (4) presence of abnormalities of the surrounding retina in all (100%) eyes. The OCT characteristics correlated well with biomicroscopic findings, and these characteristics may be predictive for final visual acuity (VA) in TMHs. Only one of the TMHs closed spontaneously in our series. CONCLUSION: Optical coherence tomography complements biomicroscopy in the evaluation of full-thickness TMHs.

Acute endophthalmitis following intravitreal bevacizumab (Avastin) injection.

PURPOSE: To report two cases of acute endophthalmitis following intravitreal bevacizumab injection. METHODS: Two patients with exudative age-related macular degeneration were treated sequentially with an intravitreal injection of bevacizumab and developed signs of severe but painless infectious endophthalmitis 2 days later. Vitreous samples were obtained, followed by the injection of vancomycin 1 mg/0.1 ml and ceftazidime 2.25 mg/0.1 ml. Pulsed-field gel electrophoresis (PFGE) was used to determine whether the isolated microorganisms were the same. RESULTS: Coagulase-negative staphylococci were identified and isolated from the vitreous specimen of both patients. PFGE revealed different patterns of banding, excluding that interpatient contamination occured. CONCLUSIONS: Infectious endophthalmitis is a potential complication of intravitreal bevacizumab injection.

Alterações oculares de doenças sistêmicas: retinopatia diabética e oftalmopatia de Graves / Systemic diseases with ocular involvement: diabetic retinopathy, Grave's disease

Das doenças sistêmicas com alterações oculares, destacam-se a retinopatia diabética e a oftalmopatia de Graves que têm em comum vários aspectos: são alterações endócrinas e metabólicas, auto-imunes e o acometimento ocular pode ser grave, levando à perda da função visual. A orientação correta do médico generalista pode modificar o prognóstico visual dos portadores destas doenças, pois dá oportunidade para que o oftalmologista intervenha no momento adequado.No diabetes a principal alteração ocular ocorre na retina, com o desenvolvimento da retinopatia diabética que se manifesta em 30% dos casos.Na oftalmopatia de Graves é importante o esclarecimento de que nem sempre as alterações oculares estão relacionadas a alterações hormonais que possam sinalizar a atividade da doença. A doença ocular pode ocorrer no hipertireoidismo, bem como no hipotireoidismo; em pacientes sem alterações hormonais afeta principalmente os tecidos adiposo e muscular da órbita e do olho. Descreve-se neste artigo o tratamento atual da retinopatia diabética e da oftalmopatia de Graves para que os pacientes possam ser orientados e esclarecidos sobre as condutas a serem adotadas pelo oftalmologista.

An intravitreal biodegradable sustained release naproxen and 5-fluorouracil system for the treatment of experimental post-traumatic proliferative vitreoretinopathy.

BACKGROUND/AIMS: To determine the potential of an intravitreal sustained release naproxen and 5-fluorouracil (NA/5-FU) codrug for the treatment of experimental proliferative vitreoretinopathy (PVR) in a model for trauma associated tractional retinal detachment (TRD). METHODS: Sustained release pellets were prepared by covalently linking naproxen to 5-fluorouracil. Drug release was tested in vitro and toxic effects were evaluated by electroretinography and light microscopy. Traumatic PVR was induced in pigmented rabbits by performing a scleral laceration, followed by repair and intravitreal injection of 0.4 ml of autologous blood. Thirty six eyes were treated with a sustained release implant containing 1.5 mg NA/5-FU as a codrug and 36 control eyes were submitted to surgery alone. Eyes were evaluated for TRD by serial indirect ophthalmoscope examination at different time points followed by postmortem fundus evaluation of the enucleated eye RESULTS: The NA/5-FU pellets were found to provide linear release of 5-FU and naproxen over the 30 day duration of the in vitro release test. Both the severity of PVR grade and the percentage of eyes with moderate or worse tractional detachment were significantly lower in eyes treated with the codrug pellet. There were no drug related toxic effects evident on histopathological or electroretinograph examination of eyes containing the NA/5-FU pellet. CONCLUSIONS: The results suggest that this NA/5-FU codrug device effectively inhibits the progression of PVR in a rabbit trauma model that closely resembles PVR in humans. Additional studies to add knowledge to these initial findings and to clarify the potential of the codrug device for the treatment of human PVR are warranted.

Indocyanine green mediated photothrombosis for the management of predominantly classic choroidal neovascularisation caused by age related macular degeneration.

AIMS: To study the effectiveness of indocyanine green mediated photothrombosis in the management of predominantly classic subfoveal choroidal neovascularisation associated with age related macular degeneration. METHODS: Prospective, non-comparative, interventional case series of nine patients with predominantly classic subfoveal choroidal neovascularisation secondary to age related macular degeneration who declined photocoagulation or verteporfin photodynamic therapy. Patients were submitted to one or more treatments with an intravenous injection of a small volume of high concentration indocyanine green solution followed by low irradiance, large spot 810 nm continuous laser application via a transpupillary approach. Main outcome measures were change in best corrected visual acuity and macular exudative manifestations. RESULTS: After 12 months of follow up, the final best corrected visual acuity was the same (plus or minus two ETDRS lines) in five eyes (55%), improved more than two ETDRS lines in three eyes (33%), and worsened by more than two lines in the remaining eye. The improved vision was probably related to partial or complete restoration of the macular architecture as a result of fluid resolution, whereas the worsened vision was primarily the result of treatment failure in achieving substantial choroidal neovascular occlusion. There were no complications related to the procedure. CONCLUSION: Indocyanine green mediated photothrombosis may be an effective alternative treatment for predominantly classic subfoveal choroidal neovascularisation caused by age related macular degeneration.

Aerobic bacterial conjunctival flora in diabetic patients.

OBJECTIVE: To study the aerobic conjunctival flora of diabetic patients and its relation to the presence and level of diabetic retinopathy and the duration of the disease. METHODS: One hundred three patients from the diabetic retinopathy screening program of the Federal University of São Paulo with no evidence of ocular surface disease were included. The diabetic patient cohort was compared with 60 nondiabetic subjects. All patients underwent slit-lamp evaluation, conjunctival scrapings, and indirect ophthalmoscopy. RESULTS: The frequency of positive conjunctival cultures was significantly higher in the diabetic group (94.18%) than in the nondiabetic group (73.33%). Among diabetic patients, a significantly higher frequency of positive cultures was detected in those with diabetic retinopathy than in those without retinopathy. Neither the duration of the diabetes nor the hypoglycemic therapy correlated with the culture results. Coagulase-negative Staphylococcus was the most common microorganism isolated, and its identification was more frequent in patients with retinopathy than in those without diabetic retinopathy. CONCLUSION: Diabetic patients have a significantly higher number of positive conjunctival cultures. The presence of diabetic retinopathy was correlated with an increase in positive cultures and a higher proportion of coagulase-negative Staphylococcus.

Indocyanine green-mediated photothrombosis combined with intravitreal triamcinolone for the treatment of choroidal neovascularization in serpiginous choroiditis.

PURPOSE: To report a case of peripapillary choroidal neovascularization (CNV) complicating serpiginous choroiditis that was treated by a single indocyanine green (ICG)-mediated photothrombosis session combined to intravitreous triamcinolone acetonide (TA) injection. METHODS: Interventional case report. A 48-year-old patient with peripapillary CNV was submitted to a laser-dye-mediated technique that uses ICG and low-intensity 810-nm light for continuous laser application; TA was then injected into the vitreous cavity 1 hour later, and prospective evaluation with fluorescein and ICG angiography as well as optical coherence tomography (OCT) was performed. RESULTS: At 2 weeks after treatment, best-corrected visual acuity improved from 20/200 to 20/50, with further improvement to 20/20-1 in the subsequent 10 weeks. Absence of fluorescein leakage from the CNV and OCT evidence of resolved retinal oedema was observed at that time. Clinical stabilization was maintained up to 1 year of follow-up. There was no significant complication related to the procedure. CONCLUSION: Combined ICG-mediated photothrombosis and intravitreous TA induced rapid and significant visual acuity recovery in this particular case of peripapillary CNV complicating serpiginous choroiditis. Accordingly, angiographic and OCT findings demonstrated neovascular lesion regression and restoration of the macular architecture.

Diode laser modifications for treatment of choroidal neovascularisation.

The aim of this paper is to describe various diode laser modifications and their use in treating choroidal neovascularisation in age-related macular degeneration. Diode lasers are used to treat selected choroidal neovascular membranes. Alterations in microprocessor connectivity, and parameters such as maximum spot size, light delivery time and coupled Joule meter, were made so that ophthalmic surgeons could specify treatment possibilities. A trimodal (photocoagulation, transpupillary thermotherapy and photodynamic therapy) application laser device coupled to a single light source has been developed. The new diode laser modifications were technically successful. Microprocessor connectivity was obtained, larger spot sizes were achieved, light delivery time could be extended and energy parameters were available at the display.

Optical coherence tomography in choroidal neovascular membrane associated with Best's vitelliform dystrophy.

A 29-year-old black male with Best's dystrophy presented an elevated choroidal neovascular membrane in the right eye that was diagnosed and followed with fluorescein-indocyanine green angiography and optical coherence tomography. The subretinal neovascularization was successfully treated with argon laser photocoagulation. One month later, the visual acuity improved and an optical coherence tomography confirmed regression of the serous macular detachment. The final clinical picture remained stable for 12 months of follow-up.

Choriocapillaris photodynamic therapy using indocyanine green.

PURPOSE: To evaluate the potential of photodynamic therapy using indocyanine green for occlusion of choroidal neovascularization, the authors studied efficiency and collateral damage of photodynamic therapy-induced photothrombosis in the rabbit choriocapillary layer. METHODS: Fundus photography, fluorescein angiography, and light and transmission electron microscopy were used to study the efficiency of photodynamic therapy-induced photothrombosis using indocyanine green as the photosensitizer, and to assess the resultant collateral damage. The delivery system consisted of a modified infrared diode laser tuned to 810 nm, near the maximum absorption peak of indocyanine green. RESULTS: Choriocapillary occlusion was achieved at indocyanine green doses of 10 and 20 mg/kg and a radiant as low as 6.3 J/cm(2). When photodynamic therapy was performed with indocyanine green doses of 10 mg/kg, damage to the neural retina was minimal. Only inner photoreceptor segments showed degeneration, probably secondary to choroidal ischemia. Bruch membrane remained intact. Retinal pigment epithelium was invariably damaged, as seen with other photosensitizers. Temporary occlusion of large choroidal vessels occurred at both dye doses. CONCLUSIONS: In this experimental study, photodynamic therapy using indocyanine green and 810-nm light irradiation produced endothelium-bound intraluminal photothrombosis, with preservation of the retinal architecture and minimal loss of visual cells. Membrane targetability, hydrophilic and fluorescent properties, and activation at 805 nm suggest indocyanine green as a potential photosensitizer for choroidal neovascularization. These combined considerations point toward further study of photodynamic therapy using indocyanine green for the treatment of choroidal vascular disease.

Optical coherence tomography evaluation of idiopathic macular hole treatment by gas-assisted posterior vitreous detachment.

PURPOSE: To report a case of idiopathic macular hole, with vitreoretinal traction confirmed by optical coherence tomography that was successfully treated by a single intravitreous perfluoropropane (C(3)F(8)) gas bubble injection. METHODS: Case report. A 65-year-old patient with idiopathic macular hole (stage 2, one eye) received an intravitreous gas injection and was prospectively followed with optical coherence tomography. RESULTS: A complete posterior vitreous detachment was achieved within 6 weeks after gas injection. Visual acuity improved from 20/80 to 20/25 by 10 months of follow-up. Optical coherence tomography disclosed vitreoretinal traction release and macular hole closure. No complications were related to the procedure. CONCLUSION: This simple procedure can assist a complete posterior vitreous detachment with relief of the hyaloid-foveolar traction, facilitating macular hole closure.

Transretinal feeder vessel ligature in von Hippel-Lindau disease.

PURPOSE: To present a new technique called transretinal feeder vessel ligature for the treatment of retinal angiomas. METHODS: Case report of a patient with peripheral retinal angiomas previously treated unsuccessfully with photocoagulation who responded to this new, alternative surgical treatment. RESULTS: The retinal angiomas decreased in size although two new feeder vessels appeared and the lesions showed a regression pattern after additional laser therapy over the vascular tumors. CONCLUSIONS: A transretinal feeder vessel ligature in association with vitrectomy and photocoagulation may be useful for some advanced or non-responsive cases of retinal angiomas.

Photodynamic therapy with indocyanine green for occult subfoveal choroidal neovascularization caused by age-related macular degeneration.

PURPOSE: To report two cases of occult subfoveal choroidal neovascularization (CNV) due to age-related macular degeneration (AMD) that were successfully treated utilizing large spot, low irradiance 810-nm light and intravenous injection of small-volume, high-concentration indocyanine green (ICG) bolus, termed ICG-mediated photodynamic therapy (i-PDT). METHODS: Case report. Two patients with occult subfoveal CNV (two eyes) due to AMD were treated with i-PDT and were prospectively followed with fluorescein and indocyanine green angiography as well as optical coherence tomography (OCT). RESULTS: Both patients presented a 3-line increase in visual acuity by twelve months of follow-up. A significant regression of the CNV was demonstrated by fluorescein angiography within one week following treatment. OCT revealed diminishing of the subretinal fluid. There were no complications related to the procedure. CONCLUSION: i-PDT induced CNV regression and improved the visual acuity in these particular cases, suggesting a new and promising way to treat neovascular AMD. The partial anatomical restoration associated to the improvement of the visual function observed in both patients suggests that a selective therapeutic effect was achieved. Further studies are required not only to assess the clinical value of this treatment but also to better understand the major mechanism of action involving this novel treatment modality.