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In vivo imaging of choroidal angiogenesis using fluorescence-labeled cationic liposomes.

Hua J, Gross N, Schulze B, Michaelis U, Bohnenkamp H, Guenzi E, Hansen LL, Martin G, Agostini HT - Mol. Vis. (2012)

Bottom Line: The best signal was obtained with CL-ICG.These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV.Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable.

View Article: PubMed Central - PubMed

Affiliation: University Eye Hospital, Albert-Ludwigs University of Freiburg, Killianstrasse 5, Freiburg im Breisgau, Germany

ABSTRACT

Purpose: Precise monitoring of active angiogenesis in neovascular eye diseases such as age-related macular degeneration (AMD) enables sensitive use of antiangiogenic drugs and reduces adverse side effects. So far, no in vivo imaging methods are available to specifically label active angiogenesis. Here, we report such a technique using fluorophore-labeled cationic liposomes (CL) detected with a standard clinical in vivo scanning laser ophthalmoscope (SLO).

Methods: C57Bl/6 mice underwent laser coagulations at day 0 (d0) to induce choroidal neovascularization (CNV). Liposomes labeled with Oregon green, rhodamine (Rh), or indocyanine green (ICG) were injected into the tail vein at various time points after laser coagulation, and their fluorescence was observed in vivo 60 min later using an SLO, or afterwards in choroidal flatmounts or cryosections.

Results: SLO detected accumulated fluorescence only in active CNV lesions with insignificant background noise. The best signal was obtained with CL-ICG. Choroidal flatmounts and cryosections of the eye confirmed the location of retained CL in CNV lesions. Neutral liposomes, in contrast, showed no accumulation.

Conclusions: These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV. This novel, non-invasive SLO imaging technique could improve risk assessment and indication for current intraocular antiangiogenic drugs in neovascular eye diseases, as well as monitor therapeutic outcomes. Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable.

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Related in: MedlinePlus

Cationic liposomes (CL) were localized in the choroidal neovascularization (CNV). At d5 or d14 after laser coagulation, CL labeled with rhodamine were applied intravenously. Sixty min later, the mice received FITC-lectin. Then, the eyes were enucleated, and cryosections were prepared. The CNV is situated between the retina and the choroid at the site where the nucleic layers (blue) of the retina are distorted. CL were found exclusively in the CNV.
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f5: Cationic liposomes (CL) were localized in the choroidal neovascularization (CNV). At d5 or d14 after laser coagulation, CL labeled with rhodamine were applied intravenously. Sixty min later, the mice received FITC-lectin. Then, the eyes were enucleated, and cryosections were prepared. The CNV is situated between the retina and the choroid at the site where the nucleic layers (blue) of the retina are distorted. CL were found exclusively in the CNV.

Mentions: To detect CL in histological sections, CL-Rh was applied intravenously, and blood vessels were labeled with fluorescein cyanate (FITC) lectin. Figure 5 shows representative images of laser-CNV at d5 and d14 after laser coagulation. The images show a well defined vessel structure in the retina and in the choroid. Staining with ToPro-3 allowed the nuclear layers of the retina that were deformed at the laser site to be identified. CL were exclusively found at the laser site. However, CL were not confined to the vessels but showed some spotty distribution around the vessels within the area of the CNV. There was no non-specific binding of CL to resting blood vessels, to photoreceptors, or to other structures. The amount of CNV labeled with CL-Rh increased from d5 to d14 confirming the results from the SLO and flatmount analysis.


In vivo imaging of choroidal angiogenesis using fluorescence-labeled cationic liposomes.

Hua J, Gross N, Schulze B, Michaelis U, Bohnenkamp H, Guenzi E, Hansen LL, Martin G, Agostini HT - Mol. Vis. (2012)

Cationic liposomes (CL) were localized in the choroidal neovascularization (CNV). At d5 or d14 after laser coagulation, CL labeled with rhodamine were applied intravenously. Sixty min later, the mice received FITC-lectin. Then, the eyes were enucleated, and cryosections were prepared. The CNV is situated between the retina and the choroid at the site where the nucleic layers (blue) of the retina are distorted. CL were found exclusively in the CNV.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351413&req=5

f5: Cationic liposomes (CL) were localized in the choroidal neovascularization (CNV). At d5 or d14 after laser coagulation, CL labeled with rhodamine were applied intravenously. Sixty min later, the mice received FITC-lectin. Then, the eyes were enucleated, and cryosections were prepared. The CNV is situated between the retina and the choroid at the site where the nucleic layers (blue) of the retina are distorted. CL were found exclusively in the CNV.
Mentions: To detect CL in histological sections, CL-Rh was applied intravenously, and blood vessels were labeled with fluorescein cyanate (FITC) lectin. Figure 5 shows representative images of laser-CNV at d5 and d14 after laser coagulation. The images show a well defined vessel structure in the retina and in the choroid. Staining with ToPro-3 allowed the nuclear layers of the retina that were deformed at the laser site to be identified. CL were exclusively found at the laser site. However, CL were not confined to the vessels but showed some spotty distribution around the vessels within the area of the CNV. There was no non-specific binding of CL to resting blood vessels, to photoreceptors, or to other structures. The amount of CNV labeled with CL-Rh increased from d5 to d14 confirming the results from the SLO and flatmount analysis.

Bottom Line: The best signal was obtained with CL-ICG.These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV.Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable.

View Article: PubMed Central - PubMed

Affiliation: University Eye Hospital, Albert-Ludwigs University of Freiburg, Killianstrasse 5, Freiburg im Breisgau, Germany

ABSTRACT

Purpose: Precise monitoring of active angiogenesis in neovascular eye diseases such as age-related macular degeneration (AMD) enables sensitive use of antiangiogenic drugs and reduces adverse side effects. So far, no in vivo imaging methods are available to specifically label active angiogenesis. Here, we report such a technique using fluorophore-labeled cationic liposomes (CL) detected with a standard clinical in vivo scanning laser ophthalmoscope (SLO).

Methods: C57Bl/6 mice underwent laser coagulations at day 0 (d0) to induce choroidal neovascularization (CNV). Liposomes labeled with Oregon green, rhodamine (Rh), or indocyanine green (ICG) were injected into the tail vein at various time points after laser coagulation, and their fluorescence was observed in vivo 60 min later using an SLO, or afterwards in choroidal flatmounts or cryosections.

Results: SLO detected accumulated fluorescence only in active CNV lesions with insignificant background noise. The best signal was obtained with CL-ICG. Choroidal flatmounts and cryosections of the eye confirmed the location of retained CL in CNV lesions. Neutral liposomes, in contrast, showed no accumulation.

Conclusions: These results establish fluorophore-labeled CL as high affinity markers to selectively stain active CNV. This novel, non-invasive SLO imaging technique could improve risk assessment and indication for current intraocular antiangiogenic drugs in neovascular eye diseases, as well as monitor therapeutic outcomes. Labeling of angiogenic vessels using CL can be of interest not only for functional imaging in ophthalmology but also for other conditions where localization of active angiogenesis is desirable.

Show MeSH
Related in: MedlinePlus