Clinical evaluation of experimentally induced choroidal neovascularizations in pigmented rabbits by subretinal injection of lipid hydroperoxide and consecutive preliminary photodynamic treatment with Tookad.

PURPOSE: Up to date several approaches have been undertaken to achieve an 'easy-to-handle' animal model of choroidal neovascularizations (CNVs) in rabbits; however, so far in none of the studies could healthy retinal tissue be maintained, which is mandatory to further investigate the effects of photodynamic therapy (PDT) or anti-vascular-endothelial-growth-factor treatments. It was our aim to reevaluate and verify the method of inducing experimental CNVs in rabbits using subretinally injected linoleic acid hydroperoxide (LHP) as proposed by Tamai et al. and to use it for experimental PDT. MATERIAL AND METHODS: In 33 eyes of Chinchilla breed rabbits LHP of two different concentrations (25 and 100 microg/50 microl) was injected into the subretinal space via a transvitreal approach under guidance of an operation microscope. Ophthalmoscopic and angiographic examinations were performed on days 3, 7, 14 and 28 after surgery. Preliminary PDT with different experimental parameter sets was performed in 3 eyes using the new photosensitizer Tookad. RESULTS: Using LHP in the higher concentration, an angiographically determined CNV induction was observed in 27% of all injection sites (n = 34) on days 14 and 28 revealing early well-demarcated and progressive leakage. No CNV was detected at the lower LHP concentration (60 injection sites). Subretinal CNV was verified histologically revealing vessel formation above the retinal pigment epithelium level. Herein, a significant damage to the outer retinal layers was always observed; however, the general structure of the choriocapillary layer was maintained. Tookad PDT was clinically able to completely stop leakage in 1 case and reduce leakage in 2 cases. Histologically the choriocapillary layer was occluded. CONCLUSION: Subretinal injection of LHP induces angiographically well-demarcated classic CNVs in rabbits; however, the CNV rate was low, and histology revealed severe damage of the outer retinal layers but not of the choriocapillary layer, which is important for studying PDT interactions. Preliminary experimental PDT could clinically stop or reduce leakage from angiographic CNV. Due to the small CNV rate and the significant collateral retinal tissue damage, this model seems to be only of partial suitability for investigating new treatment modalities in CNV.

Investigation of selective retina treatment (SRT) by means of 8 ns laser pulses in a rabbit model.

BACKGROUND: It has been shown that selective retina treatment (SRT) using a train of 1.7 microseconds laser pulses allows selective damage of the retinal pigment epithelium (RPE) while sparing the adjacent photoreceptors and thus avoiding laser scotoma. It was the purpose of this work to investigate SRT laser effects with Q-switched pulses of only 8 nanoseconds in duration by evaluating the angiographic and ophthalmoscopic damage thresholds and the damage range by histology in a rabbit model. MATERIALS AND METHODS: A flash lamp pumped frequency doubled (532 nm) Nd:YAG laser with 8 nanoseconds pulse duration was used. In total 210 laser lesions, each calculated to be 102 microm in diameter on retina, were applied through a slit lamp onto the fundus of six eyes of Chinchilla Bastard rabbits. The rabbits were irradiated with increasing energies with single pulses and a train of 10 laser pulses at 10 Hz. After treatment fundus photography and angiography were performed to determine the damage thresholds (ED(50)-probability of RPE cell damage and neurosensory retinal damage) as well as the safety range between both thresholds (ratio of angiographic ED(86) vs. ophthalmoscopic ED(14)). Selected histology was taken for single and repetitive pulse lesions after treatment. RESULTS: Angiographic and ophthalmoscopic ED(50)-thresholds decreased with increasing number of pulses. For single pulse application ophthalmoscopic and angiographic ED(50) were determined to 365 and 144 mJ/cm(2), respectively. Regarding 10 pulses 266 and 72 mJ/cm(2) were found. No retinal hemorrhages or disruptions were observed for both sets of parameters. The therapeutic window between angiographic and ophthalmoscopic threshold revealed a factor of 3.1 for single pulses and 2.3 for repetitive pulse irradiation. The safety range respectively had a factor of 0.8 (single pulses) and 1.7 (10 pulses). Histologic examination of laser lesions with single and repetitive pulses at radiant exposures within the therapeutic window-292 and 213 mJ/cm(2) respectively-revealed damaged RPE, intact Bruch's membrane and choriocapillaries. Photoreceptors were partly spared but also damaged to various extents. CONCLUSIONS: Short laser pulses of 8 nanoseconds pulse duration can damage the RPE without retinal hemorrhage or disruption. Selective damage of the RPE without affecting the photoreceptors can only rarely be achieved due to the small safety range. Thus, so far microsecond laser pulses for SRT seems favorable compared to nanosecond pulses in order to prevent unintentional photoreceptor damage.

Evaluation of the new photosensitizer Tookad (WST09) for photodynamic vessel occlusion of the choroidal tissue in rabbits.

PURPOSE: To determine the efficacy of Tookad (WST09; Negma-Lerads, Magny-Les-Hameaux, France) photodynamic therapy (T-PDT) by evaluating the angiographic and histologic closure of choroidal vessels at different radiance exposures, drug dosages, and intervals between photosensitizer injection and laser application in a rabbit model. METHODS: Chinchilla Bastard rabbits were injected intravenously with three different dye concentrations (2.5, 5, and 10 mg/kg) before application of light. In every group T-PDT was performed at four different times after injection: 5, 15, 30, and 60 minutes with different radiance exposures ranging from 200 to 3 J/cm2. Fundus photographs and fluorescein angiograms were obtained 90 minutes after injection. Follow-up angiographies were performed at days 1, 3, 7, and 14 after initial treatment. Histology was performed in selected cases immediately after treatment and on days 1, 3, and 7. RESULTS: Immediately after irradiation, most of the visible lesions were angiographically hyperfluorescent due to damaged vessel endothelium and associated RPE damage. Lesions from high-radiance exposures revealed immediate hypofluorescence, indicating vessel closure. Hypofluorescent lesions appeared mainly during day 1 (all lesions angiographically visible, some hypofluorescent) to day 3 (all lesions hypofluorescent) after treatment. At day 7, ophthalmoscopically visible hyperpigmentation took place in all lesions. ED50 thresholds for angiographic hypofluorescence determined at day 3 after treatment with 2.5 mg/kg were 18.8 J/cm2 (5 minutes), 62.0 J/cm2 (15 minutes), and >100 J/cm2 (30 minutes); with 5 mg/kg, 8.4 J/cm2 (5 minutes), 22.8 J/cm2 (15 minutes), 54.5 J/cm2 (30 minutes), and >100 J/cm2 (60 minutes); and with 10 mg/kg, 11.7 J/cm2 (30 minutes) and 54.1 J/cm2 (60 minutes). Histology of the angiographically hypofluorescent lesions revealed vessel thrombosis in all groups 1 hour after PDT up to 7 days after treatment. Sparing of photoreceptors indicated selectivity of T-PDT; however, slight damage was partly observable. After 7 days, localized proliferation of the RPE cells was noted and was enhanced 14 days after treatment. CONCLUSIONS: T-PDT has the potential to achieve selective choroidal vessel occlusion with proper parameter selection, such as (1) 2.5 mg/kg, 5 minutes, 100 J/cm2; (2) 5 mg/kg, 5 minutes, 25 J/cm2; or (3) 5 mg/kg, 15 minutes, 50 J/cm2; however, slight damage to the photoreceptors cannot be ruled out. RPE proliferation indicates primary RPE damage due to PDT, also described with the use of all other photosensitizers.