Choroidal Neovascularization In Rats Research Articles

Quantitative Image Analysis of Laser-induced Choroidal Neovascularization in Rat

Rodent models of laser-induced choroidal neovascularization (CNV) are now extensively used to identify angiogenic proteins, determine the role of specific genes with knockout mice, and evaluate the efficacy and safety of anti-angiogenic therapies. CNV is typically evaluated by fluorescein angiography or vascular endothelial cell labeling in histologic sections. The current study examined an alternative method using high molecular weight FITC-dextran (MW 2×106) for high resolution angiography in RPE-choroid-sclera flat mounts. At 24hr after lasering, the lesions appeared as a circular weakly fluorescent area of approximately equal diameter to the laser spot. No FITC-dextran labeled blood vessels were visible in the lesion at day 1. Three days after lasering, 47% of the lesions showed FITC-dextran labeling indicative of CNV. The incidence (71%) and extent of CNV increased by day 6, and by day 10 all lesions were vascularized, and the maximal area was attained. No significant change followed day 10, and the neovascular area remained constant through day 31. The highest rate of blood vessel growth (between 3 and 10 days after laser) correlates with the peak expression of VEGF, bFGF, and their receptors shown in previous studies. Morphologic analysis of flat mounts and histologic sections showed that the neovascular plexus in most lesions originates from deeper choroidal vessels in the center of the lesion, grows towards the neural retina, then branches circumferentially to anastamose with uninjured choriocapillaris. The microvessels in these lesions are broad and flat, similar to normal choriocapillaris. In a separate study, rats were treated daily with the angiostatic corticosteroid dexamethasone (20–500μ g kg−1day−1), and CNV was examined at day 10 in FITC-dextran labeled flat mounts and histologic sections. Dexamethasone dose-dependently inhibited CNV, and its highest dose inhibited approximately 95% of CNV labeled by FITC-dextran and resulted in lesions with no detectable Factor VIII immunostaining. High resolution angiography with FITC-dextran is reproducible and quantifiable, and it may accelerate the discovery of therapeutic agents that modulate choroidal neovascularization.

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

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. 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. 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. 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.

DIGITAL IMAGING OF THE POSTERIOR SEGMENT

Indirect ophthalmoscopy and slit-lamp biomicroscopy remain indispensable tools in the diagnosis of disorders of the posterior segment. Many imaging techniques have expanded our knowledge of the anatomy and physiology of the retina and choroid by providing both structural and functional information. Fluorescein angiography has become a widely accepted and routinely used technique for imaging the retina. Digital imaging systems have allowed the use of other fluorescent dyes, mainly indocyanine green (ICG), for angiography. ICG angiography has improved our ability to image the choroidal circulation. ICG serves as an adjunct to fluorescein angiography for the evaluation of choroidal neovascularization. When used in conjunction with direct clinical examination, these imaging modalities have expanded our diagnostic and therapeutic capabilities. Notable advances in fundus cameras, optics, and filters have occurred. There are at present two main categories of systems available for angiography: conventional fundus cameras and the scanning laser ophthalmoscope. Digital imaging is a modification to the conventional fundus camera that emerged in the 1990s as an alternative to photographic film angiography. Optical Coherence Tomography (OCT) is another digital imaging technique that provides structural information about the posterior segment with higher resolution than conventional systems.

Evaluation of choroidal neovascularization in age-related macular degeneration with fluorescein and indocyanine green videoangiography.

This study is performed to evaluate the fluorescein and indocyanine green (ICG) characteristics of choroidal neovascularization (CNV) in age-related macular degeneration (ARMD). 200 eyes of 101 patients were examined with digital ICG videoangiography (ICGV). 190 of the 200 eyes had macular lesions of ARMD, and CNVs were detected in 84 (44.2%) of them. Angiographies confirmed the presence of well-defined CNV in 40 (47.6%) eyes and occult CNV in 44 (52.4%) eyes. On ICG-videoangiographic examination, 6 (13.6%) of the 44 eyes with occult CNV had vascularized pigment epithelial detachment. 27 (61.4%) had a vascularized retinal pigment epithelium. Laser photocoagulation had been performed on the remaining 11 (25%) eyes. The results of this study suggest that ICGV is a valuable technique in the diagnosis and evaluation of occult CNVs and may be suggested as an additional diagnostic tool in cases with choroidal neovascular membranes.

ATX-S10を用いた光化学治療による眼内新生血管閉塞治療の可能性

Wister rats bearing corneal neovascularization, with or without pretreatment of heparin, were subjected to photodynamic therapy (PDT) with ATX-S10, and the mechanism of vascular injury was estimated by electron microscopic analysis. After laser irradiation, neovascular endotherial cells exhibited surface irregularity, swelling of mitochondria, dilatation of endoplasmic reticulum and finally dissolution of the cytoplasm. In heparinized rats where ischemic influences derived from hemostasis were excluded, we still observed swelling of mitochondria but no dissolution of the cytoplasm. These results suggested that the target site of ATX-S10 was endotherial cell surface and mitochondria and the main mechanism might be ischemia accompanied by thrombosis. Uptake of ATX-S10 in corneal neovascularization was investigated by angiography using ATX-S10, nitrogen-pulsed laser spectrofluorometry and fluorescence microscope. These results suggested that the difference in ATX-S10 accumulation between new vessels and surrounding normal tissue increased at two to four hours after dye injection and this time period was supposed to be optimal to start laser irradiation. The optimal treatment parameters for achieving selective closure of new vessels was determined. For rabbit corneal neovascularization, the optimal irradiation fluences were 107-153J/cm2 and 38.2-76.4J/cm2 with dye doses of six and twelve mg/kg, respectively. For rat choroidal neovascularization, the optimal fluence was 22J/cm2 with a dye dose of 16mg/kg. ATX-S10 is a promising new generation sensitizer which has rapid elimination from the body and little skin phototoxicity, and it appears to be useful in the treatment of ocular neovascularization.