Effects of intravitreally injected Fc fragment on rat eyes.

PURPOSE: Anti-vascular endothelial growth factor (VEGF) drugs are used to treat neovascular eye diseases. Some of these drugs contain Fc fragments (Fc), but it is unknown how their mode of action is influenced by Fc. Therefore, this study investigated the effects of Fc on rat eyes after intravitreal injection. METHODS: Eighteen Long-Evans rats were intravitreally injected with sterile, biotin-labeled rat Fc (9.1 µg in 5 µl PBS). For control, 5 µl PBS was injected in another nine rats. Animals were sacrificed between 1 and 3 days (group 1), 7 days (group 2), and 14 days (group 3) after injection. The right eyes were examined by electron microscopy (EM). The left eyes were stained by immunohistochemistry to investigate the distribution of Fc and the presence of macrophages. RESULTS: After 1 day, Fc had penetrated into the anterior chamber and the retina up to the inner nuclear layer, and was located especially in retinal vessels. High numbers of infiltrating cells were present within the vitreous, around the ciliary body, anterior chamber and inside the retina 1-3 days after Fc injection (p < 0.02 group 1 vs. control). Immunohistochemistry and EM showed that they were macrophages or granulocytes in close association with Fc. Ultrastructurally, there were effects on the blood vessels such as thrombocyte activation and fibrin formation. CONCLUSIONS: Biotin labeling is ideal for investigating the distribution of intravitreally injected proteins in ocular tissue. Fc fragments at a dose corresponding to their concentration in standard AMD treatments induced inflammation, and particularly the attraction of immune-competent cells. This may be associated with the risk of inflammation or endophthalmitis after anti-VEGF treatment, and needs further investigation.

The effects of VEGF-A-inhibitors aflibercept and ranibizumab on the ciliary body and iris of monkeys.

PURPOSE: To investigate the effects of intravitreal ranibizumab (Lucentis®) and aflibercept (Eylea®) on the ciliary body and the iris of 12 cynomolgus monkeys with regard to the fenestrations of their blood vessels. MATERIALS AND METHODS: Structural changes in the ciliary body and in the iris were investigated with light, fluorescent, and transmission electron microscopy (TEM). The latter was used to specifically quantify fenestrations of the endothelium of blood vessels after treatment with aflibercept and ranibizumab. Each of the two ciliary bodies treated with aflibercept and the two treated with ranibizumab and their controls were examined after 1 and 7 days respectively. Ophthalmological investigations including funduscopy and intraocular pressure measurements were also applied. RESULTS: Ophthalmological investigations did not reveal any changes within the groups. Both drugs reduced the VEGF concentration in the ciliary body pigmented epithelium. The structure of the ciliary body was not influenced, while the posterior pigmented epithelium of the iris showed vacuoles after aflibercept treatment. Ranibizumab was mainly concentrated on the surface layer of the ciliary epithelium, in the blood vessel walls and the lumen of some of the blood vessels, and in the cells of the epithelium of the ciliary body. Aflibercept was more concentrated in the stroma and not in the cells of the epithelium, but as with ranibizumab, also in the blood vessel walls and some of their lumina, and again on the surface layer of the epithelium. Both aflibercept-and ranibizumab-treated eyes showed a decreased number of fenestrations of the capillaries in the ciliary body compared to the untreated controls. On day 1 and day 7, aflibercept had fewer fenestrations than the ranibizumab samples of the same day. CONCLUSIONS: Both aflibercept and ranibizumab were found to reach the blood vessel walls of the ciliary body, and effectively reduced their fenestrations. Aflibercept might eliminate VEGF to a greater extent, possibly due to a higher elimination of fenestrations in a shorter time. Moreover, the vacuoles found in the iris need further research, in order to evaluate whether they carry a possible pathological potential.

Different effects of intravitreally injected ranibizumab and aflibercept on retinal and choroidal tissues of monkey eyes.

BACKGROUND: Since there is evidence that the Fc domain of antivascular endothelial growth factor drugs may cause unexpected consequences in retinal and choroidal vessels, the effects of intravitreal ranibizumab and aflibercept on monkey eyes were investigated. METHODS: Four cynomolgus monkeys were intravitreally injected with 0.5 mg of ranibizumab and another four with 2 mg of aflibercept. Two untreated monkeys served as controls. Funduscopy, fluorescein angiography (FA), spectral-domain-optical coherence tomography (SD-OCT) and measurement of intraocular pressure (IOP) were performed. The eyes were inspected by light, fluorescence and electron microscopy. The diameter of the choriocapillaris (CC) was measured by morphometry, and the areas of the CC with free haemoglobin, CC fenestrations and endothelial thickness were quantified. RESULTS: Analysis showed ranibizumab permeated the retina via intercellular clefts, whereas aflibercept was taken up by ganglion cells, cells of the inner and outer retinal layers and the retinal pigment epithelium (RPE). Stasis and haemolysis in the choriocapillaris and choroidal vessels were more frequent after aflibercept treatment, which caused hypertrophy and death of individual RPE cells. The area of the CC was significantly reduced after both drugs compared with controls, but the reduction of the CC endothelium thickness, number of fenestrations and the areas with haemolysis were more pronounced after aflibercept. CONCLUSIONS: Ranibizumab permeated the retina through intercellular spaces, whereas aflibercept was taken up by neuronal and RPE cells. Aflibercept induced protein complex formation and more haemolysis in the choriocapillaris, leading to individual RPE cell death. The clinical significance and relation of these findings to the Fc domain or to other characteristics of aflibercept remain to be investigated.