PDT to monkey CNV with ATX-S10(Na): inappropriateness of early laser irradiation for selective occlusion.

PURPOSE: There is controversy about which mode of laser irradiation, early irradiation with low-dose photosensitizer or late irradiation with high-dose, benefits the selective occlusion of choroidal neovascularization (CNV) in photodynamic therapy (PDT). In this study, using an amphiphilic photosensitizer, 13,17-bis (1-carboxypropionyl) carbamoylethyl-8-etheny-2-hydroxy-3-hydroxyiminoethylidene-2,7,12,18-tetraethyl porphyrin sodium (ATX-S10(Na); Photochemical Inc., Okayama, Japan), photodynamic and adverse effects of early irradiation on CNV-bearing monkey eyes were investigated. METHODS: Experimentally induced CNV lesions and normal retina were irradiated with a diode laser (670-nm wavelength) at a dose of 1 to 90 J/cm(2) at 1 to 19 minutes after intravenous injection of 2 mg/kg body weight of ATX-S10(Na). Vascular occlusion and CNV recurrence were evaluated by fluorescein and indocyanine green angiography and histologic analysis, until 4 weeks after irradiation. RESULTS: Of 45 different conditions, 23 did not induce CNV closure, 20 provided both CNV occlusion and retinal vessel damage, and 2 achieved selective CNV occlusion without retinal vascular injury. Recurrence of CNV was induced in 19 of 22 CNV-occluding conditions. ATX-S10(Na) angiography showed that dyes were similarly distributed between normal vessels and CNV at early time periods after injection, whereas they were preferentially accumulated in CNV after 30 minutes. CONCLUSIONS: In PDT with ATX-S10(Na), irradiation within 20 minutes of dye injection failed to induce selective CNV occlusion, probably because there is no significant difference in the biodistribution of dye between CNV and retinal vessels. It also caused frequent CNV recurrence after extensive inflammation in the irradiated retina.

Treatment parameters for selective occlusion of experimental corneal neovascularization by photodynamic therapy using a water soluble photosensitizer, ATX-S10(Na).

Time dependent change of an accumulation of an amphiphilic photosensitizer, ATX-S10(Na) on rabbit corneal neovascularization (CoNV) was evaluated by angiography using ATX-S10(Na) as a fluorescent dye on three rabbits. The angiography showed that the dye accumulated on CoNV 3-5 hr after dye injection when the dye in the iris was minimum. The results suggested 3-5 hr after might be the optimal time to start photodynamic therapy (PDT) to occlude CoNV selectively without damage to the surrounding normal tissue such as the iris. Then the optimal treatment parameters in PDT using ATX-S10(Na) for selective occlusion of the CoNV were investigated on rabbit eyes. PDT was performed with two different time intervals between dye injection and laser irradiation of a diode laser (670 nm), different laser doses and three different dye doses on 21 animals. PDT performed immediately after dye injection selectively occluded CoNV with laser irradiations from 30.6 to 38.2 J cm(-2)and a 2 mg kg(-1)dose of ATX-S10(Na), as well as with 15.3 J cm(-2)and a 6 mg kg(-1)dose. PDT performed 4 hr after dye injection with 107.0-152.8 J cm(-2)and a 6 mg kg(-1)dose, as well as with 38.2-53.5 J cm(-2)and a 12 mg kg(-1)dose was also effective. Although PDT performed either immediately or 4 hr after ATX-S10(Na) injection selectively occluded CoNV, the width of the optimal range of radiant exposures seemed wider in PDT performed 4 hr after dye injection. It is supposed that this result is associated with the difference of dye accumulation between in CoNV and in normal tissue as shown by the present angiographical findings.

A retrospective pilot study of indocyanine green enhanced diode laser photocoagulation for subfoveal choroidal neovascularization associated with age-related macular degeneration.

PURPOSE: The effectiveness and limitations of indocyanine green (ICG) enhanced diode laser photocoagulation in treating subfoveal choroidal neovascularization (CNV) associated with age-related macular degeneration (AMD) were investigated retrospectively. METHODS: Thirty-eight eyes of 37 patients with subfoveal CNV received ICG enhanced diode laser (wavelength, 805 nm) photocoagulation in our preliminary series. Nineteen eyes had classic CNV and the others had occult CNV, which was well-delineated on ICG angiography. The rate of anatomical success and functional outcomes were investigated. Factors prognostic of a final visual acuity of 0.1 or better were analyzed. The follow-up period ranged from 6 to 51 months (mean +/- SD = 26.5 +/- 14.4 months). RESULTS: Occlusion of CNV was achieved in 35 of 38 eyes (92%), and 7 eyes (18%) showed recurrence, which was occluded by retreatment in all but 1 eye. Ten eyes (26.3%) showed improvement of visual acuity; 16 (42.1%) showed no change; and in 12 eyes (31.6%) visual acuity deteriorated. Factors prognostic of a final visual acuity of 0.1 or better were good preoperative visual acuity (Mann-Whitney U-test, P =.0028), and a relatively short distance between the edge of laser burns and the center of the foveal avascular zone (unpaired t-test, P =.0285). CONCLUSION: Indocyanine green enhanced photocoagulation achieved a higher anatomical success rate but functional outcomes equal to those with argon or krypton laser photocoagulation. A controlled prospective study is necessary to prove the efficacy of this treatment.

Photodynamic therapy for corneal neovascularization using topically administered ATX-S10 (Na).

BACKGROUND AND OBJECTIVE: We investigated the effect of photodynamic therapy (PDT) using topically administered ATX-S10(Na) on corneal neovascularization (CoNV). MATERIAL AND METHODS: Rabbit eyes with induced CoNV were treated with ATX-S10(Na) eye drops (10 mg/mL) every 5 minutes, 5 to 25 times. Five to ninety minutes after topical administration, the CoNV were irradiated with a diode laser using a wavelength of 670 nm. RESULTS: The CoNV were occluded fluorescein angiographically in 7 of 16 treated eyes. The eyes having occluded, CoNV were irradiated using fluence of 510-1019 J/cm2 within 20 minutes of eye-drop administration. However, the effect was more variable than what we found using systemic administration in our previous investigation. CONCLUSIONS: Experimental CoNV was occluded by photodynamic therapy using topically administered ATX-S100(Na), suggesting this modality as a possible treatment for CoNV avoids the side effects found with systemic administration of the dye. Further efforts to improve the eye drops in terms of pH and osmotic pressure are needed to achieve increased dye accumulation.

Accumulation of photosensitizer ATX-S10 (Na) in experimental corneal neovascularization.

PURPOSE: To determine the most appropriate time for laser irradiation to produce selective occlusion of new corneal vessels by photodynamic therapy (PDT) with a new photosensitizer, ATX-S10(Na). METHODS: The time course of the plasma levels of ATX-S10(Na) and the degree of dye accumulation in the corneal neovascularization after intravenous administration was determined in rabbit eyes. Plasma concentration of ATX-S10(Na) was analyzed by a spectrophotometer. The amount of ATX-S10(Na) in the new corneal vessels was measured by nitrogen-pulsed laser spectrofluorometry. Frozen sections of neovascularized cornea and iris were observed by fluorescence microscopy. RESULTS: Plasma ATX-S10(Na) concentration was highest 5 minutes after dye injection and rapidly decreased and reached almost zero at 24 hours, indicating its prompt excretion from the body. The amount of ATX-S10(Na) in the new corneal vessels as measured by nitrogen-pulsed laser spectrofluorometry increased and reached maximal level at 2 to 4 hours. Under fluorescence microscopy, the dye was more abundantly localized in the wall of new corneal vessels than in the normal tissue at 2 to 4 hours. CONCLUSION: These results indicate that laser irradiation between 2 and 4 hours after dye injection is appropriate for selective PDT with ATX-S10(Na) for the occlusion of new corneal vessels.

Photodynamic therapy for experimental tumors using ATX-S10(Na), a hydrophilic chlorin photosensitizer, and diode laser.

ATX-S10(Na), a hydrophilic chlorin photosensitizer having an absorption maximum at 670 nm, is a candidate second-generation photosensitizer for use in photodynamic therapy (PDT) for cancer treatment. The effectiveness of PDT using ATX-S10(Na) and a diode laser for experimental tumors was evaluated in vitro and in vivo. In-vitro PDT using ATX-S10(Na) and the diode laser showed drug concentration-, laser dose- and drug exposure time-dependent cytotoxicity to various human and mouse tumor cell lines. In Meth-A sarcoma-implanted mice, optimal PDT conditions were found where tumors were completely eliminated without any toxicity. Against human tumor xenografts in nude mice, the combined use of 5 mg / kg ATX-S10(Na) and 200 J / cm(2) laser irradiation 3 h after ATX-S10(Na) administration showed excellent anti-tumor activity, and its efficacy was almost the same as that of PDT using 20 mg / kg porfimer sodium and a 100 J / cm(2) excimer dye laser 48 h after porfimer sodium injection. Microscopic observation of tumor tissues revealed that PDT using ATX-S10(Na) and the diode laser induced congestion, thrombus and degeneration of endothelial cells in tumor vessels, indicating that a vascular shutdown effect plays an important role in the anti-tumor activity of PDT using ATX-S10(Na) and the diode laser.

In vitro plasma protein binding and cellular uptake of ATX-S10(Na), a hydrophilic chlorin photosensitizer.

ATX-S10(Na), a hydrophilic chlorin photosensitizer having an absorption maximum at 670 nm, is a candidate second-generation photosensitizer for photodynamic therapy (PDT) for cancer treatment. In this study, we examined plasma protein binding, cellular uptake and subcellular targets of ATX-S10(Na) in vitro. Protein binding ratios of 50 microg / ml ATX-S10(Na) in rat, dog and human plasma were 73.0%, 87.2% and 97.7%, respectively. Gel filtration chromatography revealed that 1 mg / ml ATX-S10(Na) bound mainly to high-density lipoprotein (HDL) and serum albumin at the protein concentration of 0.4%, with binding ratios of 46% and 36%, respectively. The free form of ATX-S10(Na) was mostly incorporated into T.Tn cells, and its cellular uptake was partially but significantly inhibited by endocytosis inhibitors such as phenylarsine oxide, chloroquine, monensin and phenylglyoxal, and by chilling the cells to 4 degrees C. However, ouabain, harmaline, sodium cyanide, probenecid and aspartic acid did not influence the uptake of ATX-S10(Na), suggesting that cellular uptake of ATX-S10(Na) was not related to sodium-potassium pump activity, sodium-dependent transporter activity, mitochondrial oxidative respiration, organic anion transporter activity or aspartic acid transporter activity. By fluorescence microscopy, lysosomal localization of ATX-S10(Na) was observed in T.Tn cells. However, electron microscopic observation revealed that many subcellular organelles such as mitochondria, endoplasmic reticulum, ribosomes, Golgi complex and plasma membrane were damaged by PDT using 25 microg / ml ATX-S10(Na) soon after laser irradiation at 50 J / cm(2), and tumor necrosis was rapidly induced. This result indicated that ATX-S10(Na) was widely distributed within the cell.

Treatment parameters for the efficacy of transscleral cyclophotocoagulation in rabbits using a diode laser.

PURPOSE: To determine parameters for the efficacy of transscleral cyclophotocoagulation (TSCPC) using a diode laser. METHODS: We performed TSCPC on 74 pigmented rabbits with different exposure powers and varying number of applications, followed by clinical observation and histological examination up to 24 weeks. RESULTS: Based on observation of the clinical course, the most favorable parameters were 600 mW and 36 or 48 applications, which did not cause severe complications and sufficiently lowered intraocular pressure (IOP). Histological examination revealed coagulation of the epitheliums and stroma of the ciliary body at 600 mW. The stroma of the ciliary body was severely damaged at 900 mW. CONCLUSIONS: Transscleral cyclophotocoagulation at 600 mW with a larger number of applications than previously reported did not cause severe complications and effected greater and more lasting lowering of IOP than TSCPC with more intense coagulation and fewer applications.

Management of orbital lymphangioma using intralesional injection of OK-432.

AIM: To treat orbital lymphangioma with an intralesional injection of OK-432 (group A Streptococcus pyogenes of human origin). METHOD: A 14 year old boy had a right orbital cystic lymphangioma. The visual acuity in the eye was 20/28. In an initial treatment, 0.02 mg of OK-432, was injected into the tumour after aspiration of the fluid contents, but no effect was seen. The second treatment was performed with 0.04 mg of OK-432. RESULT: 4 months later, the lesion had totally shrunk to fibrous tissue. The side effects were fever, a local inflammatory reaction lasting 3 days, and increased intraocular pressure, which was managed by draining the fluid contents. Visual acuity improved to 20/15, and the visual field defect and restriction of eye movement seen before treatment disappeared. No recurrence was noted 1 year after treatment. CONCLUSION: An intralesional injection of OK-432 shrunk the lymphangioma without functional disturbance and scar in the facial skin. OK-432 may be useful for orbital lymphangioma, but further studies are still warranted to determine efficacy, complications, and the optimal dose for safe treatment.

Selective photodynamic effects of the new photosensitizer ATX-S10(Na) on choroidal neovascularization in monkeys.

OBJECTIVE: To determine the optimal treatment variables for photodynamic therapy (PDT) with new photosensitizer ATX-S10(Na) (13,17-bis[1-carboxypropionyl] carbamoylethyl-8-etheny-2-hydroxy-3-hydroxyiminoethyliden e-2,7,12,18-tetranethyl 6 porphyrin sodium) to induce selective occlusion of choroidal neovascularization (CNV) in nonhuman primate eyes. METHODS: Experimental CNV was induced in monkey eyes by laser photocoagulation, and PDT was performed in neovascularized and healthy eyes with different treatment variables. At 0 to 150 minutes after 4-, 8-, and 12-mg/kg of body weight intravenous injections of ATX-S10(Na), a diode laser was irradiated at the dose of 1 to 127 J/cm2 (wavelength, 670 nm). Vascular occlusion induced by PDT was evaluated using fluorescein angiography, indocyanine green angiography, and histological examination at 1 day to 4 weeks after irradiation. RESULTS: Selective occlusion of CNV without damage to healthy retinal and choroidal capillaries was achieved in the following conditions: 30 to 74 J/cm2 irradiation at 30 to 74 minutes after the 8-mg/kg injection, and 1 to 29 J/cm2 irradiation at 30 to 74 minutes or 30 to 74 J/cm2 irradiation at 75 to 150 minutes after the 12-mg/kg dye injection. Regrowth of CNV often occurred when the retina was heavily injured by excessive PDT. CONCLUSION: By using optimal treatment variables, PDT using ATX-S10(Na) induces selective occlusion of CNV in nonhuman primate eyes, providing the possibility of therapeutic application to the clinical practice. CLINICAL RELEVANCE: Occlusion of CNV without direct damage to the sensory retina is useful to preserve visual acuity in patients with exudative age-related macular degeneration. A clinical trial of PDT using ATX-S10(Na) is desirable.

A case of glaucoma with choroidal hemangioma managed by nonpenetrating trabeculectomy.

BACKGROUND: Nonpenetrating trabeculectomy was used in a patient with glaucoma complicated by diffuse choroidal hemangioma. CASE: A 12-year-old boy suffered from glaucoma with choroidal hemangioma in the left eye. Intraocular pressure was 28 mm Hg and visual acuity was 0.04. Nonpenetrating trabeculectomy was then performed. OBSERVATIONS: Postoperative intraocular pressure was controlled at around 15 mm Hg with pilocarpine hydrochloride eye drops. Visual acuity and visual field were preserved over 19 months after the operation. CONCLUSIONS: An increase in aqueous outflow resistance was considered to be the major mechanism in the rise in intraocular pressure, based on distinct dilatation and tortuosity of the episcleral blood vessels and congestion of Schlemm's canal. Therefore, construction of aqueous drainage by nonpenetrating trabeculectomy was effective. Retaining the trabecular meshwork was also considered effective in controlling complications such as choroidal hemorrhage and postoperative flat anterior chamber.

Long-term effectiveness of photodynamic therapy by using a hydrophilic photosensitizer ATX-S10(Na) against experimental choroidal neovascularization in rats.

BACKGROUND AND OBJECTIVE: We previously demonstrated that a hydrophilic photosensitizer ATX-S10 had a potent photodynamic effect. This study was designed to reveal the long-term effectiveness of photodynamic therapy (PDT) with this agent in occluding choroidal neovascularization (CNV) and its selectivity in the neovascular tissue. STUDY DESIGN/MATERIALS AND METHODS: Experimental CNV was induced by intense photocoagulation in rat eyes. Immediately or 2 hours after intravenous injection of 8 mg/kg body weight of ATX-S10(Na), a cis isomer of ATX-S10, eyes were irradiated by a diode laser at the radiance of 3.25-65.3 J/cm(2) Vascular occlusion was identified by fundus photography, fluorescein angiography, and histology at 1, 3, 7, 14, and 28 days after PDT. As controls, non-neovascular eyes were subjected to PDT and similarly analyzed. RESULTS: By using the following treatment parameters, PDT with ATX-S10(Na) successfully occluded CNV without causing occlusion of retinal capillaries for 28 days; 7.4 and 19.6 J/cm(2) immediately after dye injection and 36.7 and 65.3 J/cm(2) 2 hours after injection. Although these conditions also caused occlusion of normal choriocapillaries and mild injuries of retinal vessels, retinal pigment epithelium, and photoreceptors at 1 day, retinal vessels and pigment epithelial cells recovered from damages by 28 days. No injuries were found in the inner retina. CONCLUSION: In optimal treatment conditions, PDT with ATX-S10(Na) can induce long-term, selective occlusion of CNV without causing irreversible damages in the inner retina.

Indocyanine green angiographic features prognostic of visual outcome in the natural course of patients with age related macular degeneration.

AIMS: To determine indocyanine green (ICG) angiographic features prognostic of visual acuity loss in eyes following a natural course of exudative age related macular degeneration (AMD). METHODS: 89 eyes of 72 patients (48 men, 24 women) aged between 50 and 87 years old (mean 69.5 (SD 8.8) years) with classic and/or occult choroidal neovascularisation (CNV) were reviewed. ICG angiographic features were classified as follows: type 1, well demarcated hyperfluorescence with late ICG leakage; type 2, well demarcated hyperfluorescence with no late dye leakage; type 3, poorly demarcated hyperfluorescence; type 4, no hyperfluorescence. Follow up ranged from 6 to 67 months (mean 19.2 (11.5) months). Logistic regression analyses were performed using change of visual acuity (worse or not) as the dependent variable, and patient age, sex, characteristics of fluorescein angiography (classic or occult CNV), location of CNV, and each ICG type as the independent variables. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. RESULTS: Type 1 CNV was associated with the highest risk for visual acuity loss (OR: 7.50, CI: 1.42-39.55, p = 0.018) among the present variables. In contrast, CNV having no ICG leakage (type 2, 3, and 4), represented no significantly increased risk. CONCLUSION: Well demarcated hyperfluorescence with late ICG leakage appears to be predictive of visual acuity loss in eyes with CNV. Thus, ICG angiography may offer a useful means of predicting visual outcomes in AMD.

Selective occlusion of choroidal neovascularization by photodynamic therapy with a water-soluble photosensitizer, ATX-S10.

BACKGROUND AND OBJECTIVE: To determine the optimal treatment parameters for selective occlusion of choroidal neovascularization (CNV) by photodynamic therapy (PDT) by using the photosensitizer ATX-S10 and a diode laser (wavelength = 670 nm). MATERIALS AND METHODS: Experimental CNV was induced in rat fundi by argon laser photocoagulation. The distribution of ATX-S10 in the chorioretina was analyzed by fluorescence microscopy, and the optimal treatment parameters for selective occlusion of CNV were investigated by changing the dosage and timing of laser irradiation. CNV closure and resulting damage of the surrounding tissue were documented by fluorescein angiography and light and electron microscopies. RESULTS: Fluorescence of ATX-S10 was observed to be localized in the vascular lumen of the retina and choroid within 5 min after dye injection and increased in intensity in CNV up to 2-6 h and decreased rapidly in normal tissue. Laser irradiation with radiant exposures of 7.4 J/cm2 applied immediately after dye injection or with 22.0 J/cm2 at 2-4 h later effectively occluded the induced CNV without causing significant damage to normal retinal capillaries and large choroidal vessels. CONCLUSIONS: PDT using ATX-S10 can selectively occlude CNV. ATX-S10 is a potentially useful photosensitizer for the treatment of CNV.

[Accumulation of a photosensitizer ATX-S 10 Na (II) to experimental corneal neovascularization].

In order to determine the most appropriate time point for laser irradiation in photodynamic therapy with a new photosensitizer, ATX-S 10 Na (II), which produces selective occlusion of new vessels, we investigated the time course of plasma levels of ATX-S 10 Na (II) after intravenous administration and degree of dye accumulation in the corneal neovascularization in rabbit eyes. Plasma ATX-S 10 Na (II) concentration decreased rapidly after injection and become virtually undetectable at 24 h, indicating rapid excretion from the body. Nitrogen-pulsed laser spectrofluorometry demonstrated that the amount of ATX-S 10 Na (II) in new corneal vessels increased and reached a maximal level 2 to 4 hours after dye injection. Under a fluorescence microscope, ATX-S 10 Na (II) was localized in the wall new corneal vessels and in extravascular tissue 2 to 4 hours after dye injection. These results indicate that the appropriate time for laser irradiation in selective PDT is between 2 and 4 hours after dye injection, when a larger amount of dye is accumulated in neovasculature tissue compared to normal tissue.

Photodynamic effect of a new photosensitizer ATX-S10 on corneal neovascularization.

In order to elucidate the mechanism by which a new photosensitizer ATX-S10 causes the photodynamic effect on neovasculature, we investigated the kinetics and localization of dye accumulation in the neovascular cornea of rats after systemic administration and the development of vascular injury induced by subsequent laser irradiation, compared to those in the normal iris. Under a fluorescence microscope, the neovascular cornea always exhibited more intense fluorescence than the iris between 0.5 and 4 hr after ATX-S10 administration, indicating the preferential deposit of dye in the former tissue. The fluorescence was found inside the vascular lumen at the earliest time period and thereafter in the vascular lining cells, interstitial tissue and infiltrating neutrophils until 6 hr. As observed using light and electron microscopy, laser irradiation performed 2.5 hr after ATX-S10 injection caused extensive vascular thrombosis with endothelial destruction, which persisted for at least 3 days. The proportion of thrombosed vessels at 6 hr after laser irradiation in the neovascular cornea (64+/-5%; n=3) was significantly (P<0.01) higher than that in the normal iris (44+/-8%; n=3). In the non-thrombosed vessels from heparinized rats, in which thrombosis-related ischemic effect was excluded, mitochondrial vacuolation was the pathologic change commonly seen in the endothelial cells, pericytes and neutrophils. Morphometric analysis revealed that the mitochondria of endothelial cells in the corneal new vessels were more severely injured than those in the iris vessels. The present results indicate that ATX-S10 is a potent photosensitizer which induces photodynamic occlusion particularly of new vessels probably due to the preferential biodistribution of dye in the neovascular tissue.

Indocyanine green angiographic aspects of multiple evanescent white dot syndrome.

PURPOSE: The authors investigated the indocyanine green angiography findings of multiple evanescent white dot syndrome (MEWDS). METHODS: Four patients with MEWDS underwent examination by indocyanine green angiography, conventional ophthalmoscopy, and fluorescein angiography. RESULTS: Fundus examination showed multiple white dots in the retinal pigment epithelium of the unilateral eye of each patient. Fluorescein angiography demonstrated early hyperfluorescence corresponding to the white dots. In the early phase, indocyanine green angiography showed no abnormal signs in the large choroidal vessels, but in the late phase, hypofluorescent lesions appeared, corresponding to the white dots. The hypofluorescent dots were clustered in the posterior pole and sporadic in the peripheral region, appearing to radiate away from the optic disc or fovea. The hypofluorescent dots disappeared at the recovery stage. CONCLUSIONS: Previous fluorescein angiographic and electrophysiologic studies have demonstrated the involvement of the retinal pigment epithelium (RPE) and photoreceptors in MEWDS: The current findings on indocyanine green angiography suggest that MEWDS affects the choriocapillaris or precapillary arterioles as well as the RPE and photoreceptors, and that the lesions spread to the midperipheral region, centering on the optic disc or fovea.

Computer assisted image analysis using the subtraction method in indocyanine green angiography.

The choroidal vessels are three dimensionally distributed and very complex in their patterns. They often appear to be overlaid in indocyanine green (ICG) angiograms so it is harder to analyze ICG angiography than fluorescein angiography. When an earlier frame is subtracted from a later frame in a sequence of angiograms, the fluorescence which has increased during the time between the two frames can theoretically be demonstrated. We applied computer-assisted image subtraction methods in selected clinical cases of directly acquired digital ICG angiography to demonstrate how this method works. We used software already installed in an IMAGEnet computer system (Topcon) for image subtraction. We applied the subtraction technique in 18 cases with various diseases. When two images with a time difference of several seconds were subtracted, filling of the choriocapillaris, the neovascularization or the pathological vessels could be observed. When the images had a time difference of several minutes, intrachoroidal dye leakage could be seen more clearly. This method is very helpful for analyzing pathological changes in ICG angiography in clinical cases, when two images are selected appropriately.