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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

Accumulation of cationic and neutral liposomes was observed in flatmount preparations. After the scanning laser ophthalmoscope (SLO) images described in Figure 3 were taken, choroidal-scleral flatmounts were prepared from the mice treated with cationic liposomes (CL)-Oregon green (OG) or neutral liposomes labeled with OG. Images were taken using fluorescence microscopy. Representative ones are shown in A. They were evaluated the same way as the SLO images (B). Values for CL-OG at d10 and d14 were significantly higher compared to those of neutral liposomes. The results confirmed that the kinetics were the same as for the SLO images. Data are means obtained from five mice. Error bars indicate SEM, and asterisks indicate statistical significance (p<0.05 as compared to d1).
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f4: Accumulation of cationic and neutral liposomes was observed in flatmount preparations. After the scanning laser ophthalmoscope (SLO) images described in Figure 3 were taken, choroidal-scleral flatmounts were prepared from the mice treated with cationic liposomes (CL)-Oregon green (OG) or neutral liposomes labeled with OG. Images were taken using fluorescence microscopy. Representative ones are shown in A. They were evaluated the same way as the SLO images (B). Values for CL-OG at d10 and d14 were significantly higher compared to those of neutral liposomes. The results confirmed that the kinetics were the same as for the SLO images. Data are means obtained from five mice. Error bars indicate SEM, and asterisks indicate statistical significance (p<0.05 as compared to d1).

Mentions: The choroids of the mice that were imaged with OG-labeled liposomes were flatmounted and investigated with fluorescence microscopy. Accumulation indices were computed analogous to the procedure with SLO and are shown in Figure 4B. Similar to the SLO data in Figure 3B, there was a significantly increased accumulation of CL-OG compared to neutral liposomes labeled with OG at d10 and d14 confirming that CL bound specifically to the CNV site. The CL-OG signal was much weaker at d1 and d5, accompanied by homogeneous background staining. Flatmounts from mice treated with neutral liposomes showed background staining as well. Background staining is typically derived from tissues such as muscle showing autofluorescence at the wavelength used for detecting OG.


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)

Accumulation of cationic and neutral liposomes was observed in flatmount preparations. After the scanning laser ophthalmoscope (SLO) images described in Figure 3 were taken, choroidal-scleral flatmounts were prepared from the mice treated with cationic liposomes (CL)-Oregon green (OG) or neutral liposomes labeled with OG. Images were taken using fluorescence microscopy. Representative ones are shown in A. They were evaluated the same way as the SLO images (B). Values for CL-OG at d10 and d14 were significantly higher compared to those of neutral liposomes. The results confirmed that the kinetics were the same as for the SLO images. Data are means obtained from five mice. Error bars indicate SEM, and asterisks indicate statistical significance (p<0.05 as compared to d1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Accumulation of cationic and neutral liposomes was observed in flatmount preparations. After the scanning laser ophthalmoscope (SLO) images described in Figure 3 were taken, choroidal-scleral flatmounts were prepared from the mice treated with cationic liposomes (CL)-Oregon green (OG) or neutral liposomes labeled with OG. Images were taken using fluorescence microscopy. Representative ones are shown in A. They were evaluated the same way as the SLO images (B). Values for CL-OG at d10 and d14 were significantly higher compared to those of neutral liposomes. The results confirmed that the kinetics were the same as for the SLO images. Data are means obtained from five mice. Error bars indicate SEM, and asterisks indicate statistical significance (p<0.05 as compared to d1).
Mentions: The choroids of the mice that were imaged with OG-labeled liposomes were flatmounted and investigated with fluorescence microscopy. Accumulation indices were computed analogous to the procedure with SLO and are shown in Figure 4B. Similar to the SLO data in Figure 3B, there was a significantly increased accumulation of CL-OG compared to neutral liposomes labeled with OG at d10 and d14 confirming that CL bound specifically to the CNV site. The CL-OG signal was much weaker at d1 and d5, accompanied by homogeneous background staining. Flatmounts from mice treated with neutral liposomes showed background staining as well. Background staining is typically derived from tissues such as muscle showing autofluorescence at the wavelength used for detecting OG.

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