Sylvie Julien

Choriocapillaris breakdown precedes retinal degeneration in age-related macular degeneration

This work presents a combined light and electron microscopical approach to investigate the initial breakdown of the retinal pigment epithelium (RPE) and choriocapillaris (CC) in age-related macular degeneration (AMD). Perimacular sections of 12 dry and wet AMD eyes (82 ± 15 years) and 7 age-matched controls (75 ± 10 years) without retinal pathology were investigated. Disease progression was classified into 5 stages of retinal degeneration to investigate the concurrent CC breakdown. Special emphasis was laid on transitions where intact CC-RPE-retina complexes went over into highly atrophied areas. AMD sections showed elevated loss of photoreceptors, RPE and CC (p < 0.01), and thickened Bruchs membrane with increased basal laminar and linear deposits compared with controls. Up to 27% of the CC was lost in controls although RPE and retina were still intact. This primary loss of CC further increased with AMD (up to 100%). The data implicate that CC breakdown already occurs during normal aging and precedes degeneration of the RPE and retina with AMD, defining AMD as a vascular disease. Particular attention should be given to the investigation of early AMD stages and transitional stages to the late stage that reveal a possible sequence of degenerative steps with aging and AMD.

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.

Antipermeability and antiproliferative effects of standard and frozen bevacizumab on choroidal endothelial cells

Background: Bevacizumab is an antiangiogenic compound developed to target tumour vessels. Its off-label use in ophthalmology requires in vitro testing on ocular cells. Aim: To quantify the antipermeability and antiproliferative effects of bevacizumab on cultured choroidal endothelial cells (CECs). It was examined whether deep-freezing of bevacizumab attenuates its antiangiogenic activity. Methods: Porcine CECs were cultured in permeable insert systems. Permeability of the cell monolayers was quantified by a fluorescent isothiocyanate-dextran assay after treatment with vascular endothelial growth factor (VEGF; 20–100 ng/ml) alone and in combination with bevacizumab (0.1–1 mg/ml). Proliferation of the CECs was tested using a “wound scratch” assay. The experiments were repeated with bevacizumab after freezing at −20°C for 5 days. Results: Bevacizumab significantly reduced VEGF-induced permeability in a dose-dependant manner. A molar ratio of 2.6:1 of bevacizumab to VEGF was required for complete blocking of VEGF-induced rise in permeability. CEC proliferation was significantly blocked by bevacizumab (0.5 mg/ml). Thawed bevacizumab after deep freezing showed a moderate, but not statistically significant loss in activity. Conclusion: Bevacizumab significantly reduces VEGF-induced permeability and proliferation of CECs. Freezing and thawing of bevacizumab will affect its biological activity.

BEVACIZUMAB (AVASTIN) DOES NOT HARM RETINAL FUNCTION AFTER INTRAVITREAL INJECTION AS SHOWN BY ELECTRORETINOGRAPHY IN ADULT MICE

Purpose: Scavenging of VEGF by specific antibodies is a promising way to treat ocular conditions connected with neovascularization. Intravitreal injections of Avastin (bevacizumab) are performed frequently as a treatment of such conditions. In this study, the authors examine whether the retinal function in wild-type mice is affected by an intravitreal injection of Avastin. Methods: Electroretinography was performed in four different experimental groups of wild-type C57BL/6 mice before treatment and 1, 4, 12, and 25 days afterwards. The first group was injected intravitreally with BSS, the second one received injections of a vehicle solution, and the third group was injected with the commercial Avastin solution. In a fourth group, sham surgery was performed. Immunohistochemistry was performed in some eyes to evaluate penetration of the bevacizumab molecule through the retina. Results: In all four groups, a similar behavior of the ERG parameters could be detected. One day after the injections, the amplitudes showed a clear decrease. Later on, they recovered gradually. No difference could be seen between eyes injected with Avastin or vehicle solution. Bevacizumab immunoreactivity was already present in the whole retina half an hour after the intravitreal injection and was not detectable 25 days later. Moreover, binding of bevacizumab to endogenous mouse VEGF could be shown. Conclusions: Based on the electroretinographic findings, the authors conclude that bevacizumab does not have any toxic effects on the mouse retina and its function. The bevacizumab molecule penetrates the retina quickly. Therefore, it can act safely and very quickly, also in deeper retinal layers after its injection.

Implantation of ultrathin, biofunctionalized polyimide membranes into the subretinal space of rats.

Subretinal implants aim to replace the photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli in the subretinal space. Critical obstacles in the design of high-resolution subretinal implants include the proximity of stimulating electrodes to the target cells and enabling nutrient flow between the retina and the choroid. The present work evaluates the adhesion, migration and survival of retinal cells on an ultrathin (5 μm), highly porous (Ø 1 μm spaced 3 μm), gelatin-coated polyimide (PI) membrane. The biocompatibility was examined in mice indicating a good tolerance upon subcutaneous implantation with only a mild inflammatory response. In addition, organotypic cultures of rat retina evidenced that the porous membrane allowed the necessary nutrient flow for the retinal cell survival and maintenance. A transscleral implantation technique was applied to position the membrane into the subretinal space of rats. The effect on the obtained retinal integration was investigated in vivo using scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). In 12 out of 18 rat eyes, the implant was successfully placed subretinally. SLO and OCT demonstrated complete retinal attachment and fluorescein angiography showed no retinal vascular abnormalities over and around the implant, immediately after and up to four weeks after the implantation. Histological examination of the eyes showed a close attachment of a thin fibrocyte layer to the implant, the occlusion of the pores by living cells and the survival of some photoreceptors at the implantation site.

Loss of retinal function in aged DBA/2J mice – New insights into retinal neurodegeneration

The DBA/2J mouse is a common animal model of glaucoma. The intraocular pressure increases with age, and retinal ganglion cells (RGC) degenerate, usually starting at an age of approximately six months. In this study, we used two-year-old DBA/2J mice presuming an end-point of RGC degeneration. We investigated visual function in these animals using electroretinography (ERG) and visual evoked potentials (VEP), and we checked the number of remaining RGC by retrograde staining. Almost no RGC were left in the retina, and VEP were hardly recordable. Surprisingly, also ERG amplitudes of scotopic a-waves and b-waves, photopic b-waves and oscillatory potentials were decreased significantly by approximately 40% compared to amplitudes measured in age-matched C57BL/6J mice. The latencies were not changed in DBA/2J mice compared to C57BL/6J mice, and so were the ratios between amplitudes of a-waves, b-waves and oscillatory potentials. Our results indicate that, in addition to degeneration of RGC, also photoreceptors are affected by pathological processes in the eye caused by the mutations present in DBA/2J mice.

Lipofuscin can be eliminated from the retinal pigment epithelium of monkeys

Lipofuscin is a cytologic hallmark of aging in metabolically active postmitotic cells including neurons, cardiac muscle cells, and the retinal pigment epithelium (RPE). High levels of lipofuscin are involved in the pathogenesis of age-related macular degeneration (AMD), the main cause of blindness in the elderly population in the western world. Degradation and exocytosis of lipofuscin by RPE cells have not been observed in vivo until now, and no drug is known to eliminate the intracellular amount of lipofuscin. Here, we show that in monkeys treated with a small molecule belonging to the tetrahydropyridoethers class (n = 36 of 48 monkeys), RPE cells significantly release lipofuscin. In 4 eyes, macrophages were detected which had taken up lipofuscin. They were located between the Bruchs membrane and the RPE, and in the choroid. The quantification of pigment granules was performed by transmission electron microscopy. Our findings open the way to develop therapeutic strategies to remove lipofuscin from RPE cells, which may have implications for the treatment of age-related macular degeneration in which lipofuscin accumulation in cells is a causative factor.

Zinc deficiency leads to lipofuscin accumulation in the retinal pigment epithelium of pigmented rats.

Background Age-related macular degeneration (AMD) is associated with lipofuscin accumulation whereas the content of melanosomes decreases. Melanosomes are the main storage of zinc in the pigmented tissues. Since the elderly population, as the most affected group for AMD, is prone to zinc deficit, we investigated the chemical and ultrastructural effects of zinc deficiency in pigmented rat eyes after a six-month zinc penury diet. Methodology/Principal Findings Adult Long Evans (LE) rats were investigated. The control animals were fed with a normal alimentation whereas the zinc-deficiency rats (ZD-LE) were fed with a zinc deficient diet for six months. Quantitative Energy Dispersive X-ray (EDX) microanalysis yielded the zinc mole fractions of melanosomes in the retinal pigment epithelium (RPE). The lateral resolution of the analysis was 100 nm. The zinc mole fractions of melanosomes were significantly smaller in the RPE of ZD-LE rats as compared to the LE control rats. Light, fluorescence and electron microscopy, as well as immunohistochemistry were performed. The numbers of lipofuscin granules in the RPE and of infiltrated cells (Ø>3 µm) found in the choroid were quantified. The number of lipofuscin granules significantly increased in ZD-LE as compared to control rats. Infiltrated cells bigger than 3 µm were only detected in the choroid of ZD-LE animals. Moreover, the thickness of the Bruchs membrane of ZD-LE rats varied between 0.4–3 µm and thin, rangy ED1 positive macrophages were found attached at these sites of Bruchs membrane or even inside it. Conclusions/Significance In pigmented rats, zinc deficiency yielded an accumulation of lipofuscin in the RPE and of large pigmented macrophages in the choroids as well as the appearance of thin, rangy macrophages at Bruchs membrane. Moreover, we showed that a zinc diet reduced the zinc mole fraction of melanosomes in the RPE and modulated the thickness of the Bruchs membrane.

Immunohistochemical localisation of intravitreally injected bevacizumab in the anterior chamber angle, iris and ciliary body of the primate eye

Aims: To locate bevacizumab in the tissues related to neovascularisation in the anterior segment within 1–14 days after intravitreal injection in the primate eye. Methods: Four cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab. Control eyes remained untreated. The eyes were enucleated on day 1, 4 and 14 for immunohistochemistry, using donkey anti-human Cy3-IgG. Results: Immunoreactivity for bevacizumab was found in the blood vessels walls of the iris, anterior chamber angle and ciliary body. In the iris and chamber angle, immunoreactivity was most prominent on day 1 after injection and diminished until day 14. In the ciliary body, staining was most intense on day 4 and remained prominent until day 14. Immunoreactivity was also present in certain vessel lumens, especially in the ciliary body and the iris on day 4 and 14. Conclusion: Bevacizumab penetrates quickly into the iris, anterior chamber angle and ciliary body after intravitreal injection in the primate eye and accumulates particularly in blood-vessel walls. The highest concentration of bevacizumab in these tissues is present on day 1–4, the iris and anterior chamber angle being penetrated slightly earlier than the ciliary body. Our findings support the clinically observed rapid effect in the treatment of iris neovascularisation.

Immunohistochemical localisation of intravitreally injected bevacizumab at the posterior pole of the primate eye: implication for the treatment of retinal vein occlusion

Aim: To locate bevacizumab in the posterior pole within 1–14 days after intravitreal injection in the primate eye. Methods: Four Cynomolgus monkeys received an intravitreal injection of 1.25 mg of bevacizumab. The eyes were enucleated on days 1, 4, 7 and 14 for immunohistochemistry using donkey anti-human Cy3-IgG. Control eyes remained untreated. Results: In the optic nerve, immunoreactivity for bevacizumab was most prominent on day 1 after injection and diminished rapidly. In the blood vessels of the nerve fibre layer, the staining was intense in the walls and weak in the lumen from day 1 to 4, and was only localised in the lumen thereafter. In the macula, an accumulation of bevacizumab was observed 1 day after injection in the nerve fibre layer, the ganglion cell layer and in the photoreceptors at the level of the outer nuclear layer in the fovea centralis. Conclusion: Bevacizumab penetrates quickly into the macula, the retinal veins and the optic nerve after intravitreal injection in the primate eye, and accumulates preferentially and specifically on the vessel walls and inside the photoreceptors localised in the fovea centralis 1 day after injection. Our finding supports the clinically observed rapid effect in the treatment of retinal vein occlusion and macular oedema.