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A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye.

Sim DA, Chu CJ, Selvam S, Powner MB, Liyanage S, Copland DA, Keane PA, Tufail A, Egan CA, Bainbridge JW, Lee RW, Dick AD, Fruttiger M - Dis Model Mech (2015)

Bottom Line: We found that in vivo intravenous administration failed to label any leukocytes, whereas depot injection, either intraperitoneal or subcutaneous, was successful in labelling leukocytes infiltrating into the retina.The translation of this approach into clinical practice would enable visualization of immune cells in situ.This will not only provide a greater understanding of pathogenesis, monitoring and assessment of therapy in many human ocular diseases but might also open the ability to image immunity live for neurodegenerative disorders, cardiovascular disease and systemic immune-mediated disorders.

View Article: PubMed Central - PubMed

Affiliation: NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK University College London, Institute of Ophthalmology, London EC1V 9EL, UK.

No MeSH data available.


Related in: MedlinePlus

In vivo labelling of infiltrating leukocytes in two murine models of ocular inflammation. (A) Inflammatory infiltration of the deep retina/choroid of an eye with endotoxin-induced uveitis, imaged using a scanning laser ophthalmoscope with a near-infrared filter (790-nm diode excitation laser and 800-nm long-pass filter). ICG-labelled cells were visualized as white dots throughout the 55° field of view after an intraperitoneal (i.p.) injection of ICG (5 days before imaging) and induction of systemic inflammation with an i.p. injection of lipopolysaccharide (2 days before imaging). (B) An image of the deep retina/choroid of the same mouse was taken in the fluorescein angiography (AF) channel, using a blue-light filter (488-nm solid-state excitation laser and 500-nm long-pass filter). No white dots are present, demonstrating that white dots imaged in A are not a consequence of autofluorescence but ICG-labelled cells. (C) A control mouse that only received i.p. ICG (3 days before imaging). Imaging with a near-infrared filter showed only a few sporadic ICG-labelled cells, suggesting a low-level circulation of myeloid cells into the retina. (D) An image of the deep retina/choroid of the same mouse was obtained using the 488-nm channel, illustrating that the identified cells were not autofluorescent in this range. (E) Inflammation of a retinal vein (vasculitis) is visualized using the near-infrared filter in an eye at peak experimental autoimmune uveitis. ICG-labelled cells were visualized as white dots clustering around a segment of vasculitis. Mice with experimental autoimmune uveitis received an injection of i.p. ICG 3 days before imaging peak disease on day 26. (F) An infrared-reflectance (IR) image (820-diode excitation laser, no barrier filter) of the same mouse was obtained, which demonstrates the segment of retinal vein affected by vasculitis with increased reflectance (higher white intensity) of the vein itself, and surrounding tissues. Of note, no white dots were observed in this image, indicating that again, the white dots observed in (C) are not the result of autofluorescence but of ICG-labelled cells.
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DMM019018F3: In vivo labelling of infiltrating leukocytes in two murine models of ocular inflammation. (A) Inflammatory infiltration of the deep retina/choroid of an eye with endotoxin-induced uveitis, imaged using a scanning laser ophthalmoscope with a near-infrared filter (790-nm diode excitation laser and 800-nm long-pass filter). ICG-labelled cells were visualized as white dots throughout the 55° field of view after an intraperitoneal (i.p.) injection of ICG (5 days before imaging) and induction of systemic inflammation with an i.p. injection of lipopolysaccharide (2 days before imaging). (B) An image of the deep retina/choroid of the same mouse was taken in the fluorescein angiography (AF) channel, using a blue-light filter (488-nm solid-state excitation laser and 500-nm long-pass filter). No white dots are present, demonstrating that white dots imaged in A are not a consequence of autofluorescence but ICG-labelled cells. (C) A control mouse that only received i.p. ICG (3 days before imaging). Imaging with a near-infrared filter showed only a few sporadic ICG-labelled cells, suggesting a low-level circulation of myeloid cells into the retina. (D) An image of the deep retina/choroid of the same mouse was obtained using the 488-nm channel, illustrating that the identified cells were not autofluorescent in this range. (E) Inflammation of a retinal vein (vasculitis) is visualized using the near-infrared filter in an eye at peak experimental autoimmune uveitis. ICG-labelled cells were visualized as white dots clustering around a segment of vasculitis. Mice with experimental autoimmune uveitis received an injection of i.p. ICG 3 days before imaging peak disease on day 26. (F) An infrared-reflectance (IR) image (820-diode excitation laser, no barrier filter) of the same mouse was obtained, which demonstrates the segment of retinal vein affected by vasculitis with increased reflectance (higher white intensity) of the vein itself, and surrounding tissues. Of note, no white dots were observed in this image, indicating that again, the white dots observed in (C) are not the result of autofluorescence but of ICG-labelled cells.

Mentions: The first model was endotoxin-induced uveitis induced by systemic delivery of LPS from Escherichia coli. A previous study detected Acridine Orange-labelled leukocytes in the subretinal space/deep retina, 2 days after LPS injection (Miyahara et al., 2004). We found that ICG-labelled cells were detectable in the retina in vivo using fluorescence scanning laser ophthalmoscopy, 24 h after LPS injection, with a peak at 2 days after LPS administration (and 5 days after i.p. ICG; Fig. 3A). No signal was seen in the fluorescein channel (488 nm solid-state excitation laser and 500 nm barrier filter), indicating that the ICG signal did not derive from autofluorescence (Fig. 3B). Control animals that were injected only with ICG but not with LPS showed only a few sporadic ICG+ cells (Fig. 3C,D). This suggests low levels of peripheral cell trafficking in the normal retina, as previously inferred via flow cytometric analysis (Boldison et al., 2014; Chu et al., 2013), which dramatically increases after LPS stimulation.Fig. 3.


A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye.

Sim DA, Chu CJ, Selvam S, Powner MB, Liyanage S, Copland DA, Keane PA, Tufail A, Egan CA, Bainbridge JW, Lee RW, Dick AD, Fruttiger M - Dis Model Mech (2015)

In vivo labelling of infiltrating leukocytes in two murine models of ocular inflammation. (A) Inflammatory infiltration of the deep retina/choroid of an eye with endotoxin-induced uveitis, imaged using a scanning laser ophthalmoscope with a near-infrared filter (790-nm diode excitation laser and 800-nm long-pass filter). ICG-labelled cells were visualized as white dots throughout the 55° field of view after an intraperitoneal (i.p.) injection of ICG (5 days before imaging) and induction of systemic inflammation with an i.p. injection of lipopolysaccharide (2 days before imaging). (B) An image of the deep retina/choroid of the same mouse was taken in the fluorescein angiography (AF) channel, using a blue-light filter (488-nm solid-state excitation laser and 500-nm long-pass filter). No white dots are present, demonstrating that white dots imaged in A are not a consequence of autofluorescence but ICG-labelled cells. (C) A control mouse that only received i.p. ICG (3 days before imaging). Imaging with a near-infrared filter showed only a few sporadic ICG-labelled cells, suggesting a low-level circulation of myeloid cells into the retina. (D) An image of the deep retina/choroid of the same mouse was obtained using the 488-nm channel, illustrating that the identified cells were not autofluorescent in this range. (E) Inflammation of a retinal vein (vasculitis) is visualized using the near-infrared filter in an eye at peak experimental autoimmune uveitis. ICG-labelled cells were visualized as white dots clustering around a segment of vasculitis. Mice with experimental autoimmune uveitis received an injection of i.p. ICG 3 days before imaging peak disease on day 26. (F) An infrared-reflectance (IR) image (820-diode excitation laser, no barrier filter) of the same mouse was obtained, which demonstrates the segment of retinal vein affected by vasculitis with increased reflectance (higher white intensity) of the vein itself, and surrounding tissues. Of note, no white dots were observed in this image, indicating that again, the white dots observed in (C) are not the result of autofluorescence but of ICG-labelled cells.
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Related In: Results  -  Collection

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DMM019018F3: In vivo labelling of infiltrating leukocytes in two murine models of ocular inflammation. (A) Inflammatory infiltration of the deep retina/choroid of an eye with endotoxin-induced uveitis, imaged using a scanning laser ophthalmoscope with a near-infrared filter (790-nm diode excitation laser and 800-nm long-pass filter). ICG-labelled cells were visualized as white dots throughout the 55° field of view after an intraperitoneal (i.p.) injection of ICG (5 days before imaging) and induction of systemic inflammation with an i.p. injection of lipopolysaccharide (2 days before imaging). (B) An image of the deep retina/choroid of the same mouse was taken in the fluorescein angiography (AF) channel, using a blue-light filter (488-nm solid-state excitation laser and 500-nm long-pass filter). No white dots are present, demonstrating that white dots imaged in A are not a consequence of autofluorescence but ICG-labelled cells. (C) A control mouse that only received i.p. ICG (3 days before imaging). Imaging with a near-infrared filter showed only a few sporadic ICG-labelled cells, suggesting a low-level circulation of myeloid cells into the retina. (D) An image of the deep retina/choroid of the same mouse was obtained using the 488-nm channel, illustrating that the identified cells were not autofluorescent in this range. (E) Inflammation of a retinal vein (vasculitis) is visualized using the near-infrared filter in an eye at peak experimental autoimmune uveitis. ICG-labelled cells were visualized as white dots clustering around a segment of vasculitis. Mice with experimental autoimmune uveitis received an injection of i.p. ICG 3 days before imaging peak disease on day 26. (F) An infrared-reflectance (IR) image (820-diode excitation laser, no barrier filter) of the same mouse was obtained, which demonstrates the segment of retinal vein affected by vasculitis with increased reflectance (higher white intensity) of the vein itself, and surrounding tissues. Of note, no white dots were observed in this image, indicating that again, the white dots observed in (C) are not the result of autofluorescence but of ICG-labelled cells.
Mentions: The first model was endotoxin-induced uveitis induced by systemic delivery of LPS from Escherichia coli. A previous study detected Acridine Orange-labelled leukocytes in the subretinal space/deep retina, 2 days after LPS injection (Miyahara et al., 2004). We found that ICG-labelled cells were detectable in the retina in vivo using fluorescence scanning laser ophthalmoscopy, 24 h after LPS injection, with a peak at 2 days after LPS administration (and 5 days after i.p. ICG; Fig. 3A). No signal was seen in the fluorescein channel (488 nm solid-state excitation laser and 500 nm barrier filter), indicating that the ICG signal did not derive from autofluorescence (Fig. 3B). Control animals that were injected only with ICG but not with LPS showed only a few sporadic ICG+ cells (Fig. 3C,D). This suggests low levels of peripheral cell trafficking in the normal retina, as previously inferred via flow cytometric analysis (Boldison et al., 2014; Chu et al., 2013), which dramatically increases after LPS stimulation.Fig. 3.

Bottom Line: We found that in vivo intravenous administration failed to label any leukocytes, whereas depot injection, either intraperitoneal or subcutaneous, was successful in labelling leukocytes infiltrating into the retina.The translation of this approach into clinical practice would enable visualization of immune cells in situ.This will not only provide a greater understanding of pathogenesis, monitoring and assessment of therapy in many human ocular diseases but might also open the ability to image immunity live for neurodegenerative disorders, cardiovascular disease and systemic immune-mediated disorders.

View Article: PubMed Central - PubMed

Affiliation: NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK University College London, Institute of Ophthalmology, London EC1V 9EL, UK.

No MeSH data available.


Related in: MedlinePlus