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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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Kinetics of accumulation of cationic liposomes (CL)- indocyanine green (ICG) or CL-Oregon green (OG) was observed with the SLO in vivo. A: ICG-CL were injected at d14, and scanning laser ophthalmoscope (SLO) images were recorded in a single choroidal neovascularization (CNV). While images taken before injection of ICG (0 min) showed no signal, the ICG fluorescence (795 nm excitation, 830 nm emission) became detectable in the CNV after 10 min. Maximal intensity was observed between 30 min and 90 min with a slow decrease afterwards. Other parts of the fundus were not specifically stained. B: OG-CL were injected at d10, and SLO images were recorded in a single CNV. This series shows the early signal in the capillaries 2–3 min after injection that disappears quickly [8] while the OG-CL signal in the CNV is coming up later and for a longer time as shown in A. C: ICG-CL (40%) and CL-OG were mixed and injected at d14, and SLO images were recorded in a single CNV. Note that the OG signal is somewhat weaker. IR: CNV lesion in infrared modus.
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f2: Kinetics of accumulation of cationic liposomes (CL)- indocyanine green (ICG) or CL-Oregon green (OG) was observed with the SLO in vivo. A: ICG-CL were injected at d14, and scanning laser ophthalmoscope (SLO) images were recorded in a single choroidal neovascularization (CNV). While images taken before injection of ICG (0 min) showed no signal, the ICG fluorescence (795 nm excitation, 830 nm emission) became detectable in the CNV after 10 min. Maximal intensity was observed between 30 min and 90 min with a slow decrease afterwards. Other parts of the fundus were not specifically stained. B: OG-CL were injected at d10, and SLO images were recorded in a single CNV. This series shows the early signal in the capillaries 2–3 min after injection that disappears quickly [8] while the OG-CL signal in the CNV is coming up later and for a longer time as shown in A. C: ICG-CL (40%) and CL-OG were mixed and injected at d14, and SLO images were recorded in a single CNV. Note that the OG signal is somewhat weaker. IR: CNV lesion in infrared modus.

Mentions: The distribution of CL labeled with ICG or OG in the fundus was followed by SLO after intravenous injection. Figure 2 shows representative images at various time points after application of labeled CL-ICG or CL-OG 14 days after laser coagulation. No specific signal was detected before CL-ICG or CL-OG was injected. Circulation of CL formulations in the blood plasma was faintly visible for 2–3 min after application based on the respective fluorescence signal (Figure 2B). After that, fast accumulation of fluorescence at the CNV site was observed. Typically, the signal due to CL-OG was visible between 20 and 120 min after application. The CL-ICG signal was stronger and therefore was visible as early as 5 min after application and was observed for several hours, in some cases even for 24 h. The CL-ICG and CL-OG peak signal was observed between 30 and 90 min so that a measuring time point of 60 min after injection was chosen in the following experiments. CL-ICG resulted in a more intense and precise image compared to CL-OG (Figure 2C). In contrast, neutral liposomes did not accumulate at the laser site but remained much longer in circulation (Figure 3) similar to ICG or fluorescein in angiography.


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)

Kinetics of accumulation of cationic liposomes (CL)- indocyanine green (ICG) or CL-Oregon green (OG) was observed with the SLO in vivo. A: ICG-CL were injected at d14, and scanning laser ophthalmoscope (SLO) images were recorded in a single choroidal neovascularization (CNV). While images taken before injection of ICG (0 min) showed no signal, the ICG fluorescence (795 nm excitation, 830 nm emission) became detectable in the CNV after 10 min. Maximal intensity was observed between 30 min and 90 min with a slow decrease afterwards. Other parts of the fundus were not specifically stained. B: OG-CL were injected at d10, and SLO images were recorded in a single CNV. This series shows the early signal in the capillaries 2–3 min after injection that disappears quickly [8] while the OG-CL signal in the CNV is coming up later and for a longer time as shown in A. C: ICG-CL (40%) and CL-OG were mixed and injected at d14, and SLO images were recorded in a single CNV. Note that the OG signal is somewhat weaker. IR: CNV lesion in infrared modus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Kinetics of accumulation of cationic liposomes (CL)- indocyanine green (ICG) or CL-Oregon green (OG) was observed with the SLO in vivo. A: ICG-CL were injected at d14, and scanning laser ophthalmoscope (SLO) images were recorded in a single choroidal neovascularization (CNV). While images taken before injection of ICG (0 min) showed no signal, the ICG fluorescence (795 nm excitation, 830 nm emission) became detectable in the CNV after 10 min. Maximal intensity was observed between 30 min and 90 min with a slow decrease afterwards. Other parts of the fundus were not specifically stained. B: OG-CL were injected at d10, and SLO images were recorded in a single CNV. This series shows the early signal in the capillaries 2–3 min after injection that disappears quickly [8] while the OG-CL signal in the CNV is coming up later and for a longer time as shown in A. C: ICG-CL (40%) and CL-OG were mixed and injected at d14, and SLO images were recorded in a single CNV. Note that the OG signal is somewhat weaker. IR: CNV lesion in infrared modus.
Mentions: The distribution of CL labeled with ICG or OG in the fundus was followed by SLO after intravenous injection. Figure 2 shows representative images at various time points after application of labeled CL-ICG or CL-OG 14 days after laser coagulation. No specific signal was detected before CL-ICG or CL-OG was injected. Circulation of CL formulations in the blood plasma was faintly visible for 2–3 min after application based on the respective fluorescence signal (Figure 2B). After that, fast accumulation of fluorescence at the CNV site was observed. Typically, the signal due to CL-OG was visible between 20 and 120 min after application. The CL-ICG signal was stronger and therefore was visible as early as 5 min after application and was observed for several hours, in some cases even for 24 h. The CL-ICG and CL-OG peak signal was observed between 30 and 90 min so that a measuring time point of 60 min after injection was chosen in the following experiments. CL-ICG resulted in a more intense and precise image compared to CL-OG (Figure 2C). In contrast, neutral liposomes did not accumulate at the laser site but remained much longer in circulation (Figure 3) similar to ICG or fluorescein in angiography.

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