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

Abstract

We have developed a method to label and image myeloid cells infiltrating the mouse retina and choroid in vivo, using a single depot injection of indocyanine green dye (ICG). This was demonstrated using the following ocular models of inflammation and angiogenesis: endotoxin-induced uveitis, experimental autoimmune uveoretinitis and laser-induced choroidal neovascularization model. A near-infrared scanning ophthalmoscope was used for in vivo imaging of the eye, and flow cytometry was used on blood and spleen to assess the number and phenotype of labelled cells. ICG was administered 72 h before the induction of inflammation to ensure clearance from the systemic circulation. 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. Progression of inflammation in the retina could be traced over a period of 14 days following a single depot injection of ICG. Additionally, bright-field microscopy, spectrophotometry and flow cytometric analysis suggest that the predominant population of cells stained by ICG are circulating myeloid cells. 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.

Keywords: In vivo imaging; Indocyanine green; Inflammation.

Fig. 1.

In vitro labelling of peripheral blood mononuclear cells (PBMCs) and splenocytes in humans and mice. (A,B) Blood smear of human blood incubated in a concentration of 6.25 µg/ml indocyanine green dye (ICG) for 30 min at room temperature. A small proportion of cells stained with ICG were visualized on the near-infrared channel at 10× magnification (A) and 20× magnification (B). (C,D) Detection of ICG-stained human PBMCs by flow cytometry in cells incubated in PBS and 6.25 µg/ml of ICG reveals that 4.9% of all cells were labelled and, of these, 4.4% were CD45-high. (E,F) In mouse PBMCs, although ICG labelling was also observed in the same in vitro conditions, a smaller proportion of ICG-labelled cells were detected; 0.8% of total cells. (G,H) Mouse splenocytes were more readily labelled with ICG, with 9.5% of total cells staining with ICG.

Fig. 2.

ICG labels macrophages by internalization of the dye but does not appear to cause activation. (A) In vitro-labelled bone-marrow-derived macrophages (BMDMs, red) appear in flow cytometry more strongly labelled than CD4⁺ lymphocytes (blue; unlabelled cells are indicated by stippled lines). (B) Bright-field microscopy of BMDMs cultured for 2 h with 0.2 mg/ml ICG identifies regions of visible green dye apparently within cells. (C,D) Applying LPS, 5 ng/ml LPS only (C) and LPS+ICG (D) did not lead to a marked difference in ICG uptake. (E) There is no evidence of classical activation, as measured by the production of interleukin-6 (IL-6) from supernatants taken at different time points. Data were combined from two separate experiments (means+s.d. are shown). (F) BMDMs were incubated with 0.2 mg/ml ICG. Following several washes, PBS was incubated with the cells for 5 min, then tested by spectrophotometry and compared with supernatants from the same cells after lysis with 2% Triton. Increasing the duration of ICG incubation led to increased absorption, consistent with the release of progressive internalized ICG.

Fig. 3.

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.

Fig. 4.

In vivo labelling of infiltrating leukocytes in a laser-induced choroidal neovascularization (CNV) murine model. The scanning laser ophthalmoscope with a near-infrared filter (790-nm diode excitation laser and 800-nm long-pass filter) was used to image the retina and choroid. (A) A deep retinal/choroidal image of a control animal that did not receive an i.p. injection of ICG. No fluorescence was detected using the near-infrared filter. (B) A deep retinal/choroidal image of an animal that did receive i.p. ICG and laser induction of CNV, showing fluorescence in the retinal vessels and laser lesions. No obvious leakage of ICG could be seen in the surrounding retinal tissues. (C) A deep retinal/choroidal image of an animal that received i.p. ICG (10 days before imaging) and laser induction of CNV (7 days before imaging), showing an accumulation of ICG-labelled cells in and around the laser lesions. (C′) Magnified image of laser lesion and surrounding cells. (D) A deep retinal/choroidal image of the same animal shown in C 3 days later (13 days after i.p. ICG), showing that the intensity of the ICG signal has reduced. (D′) Magnified image of laser lesion and surrounding cells. (E) A deep retinal/choroidal image of an animal that received i.p. ICG (10 days before imaging) with six laser-induced CNV lesions (7 days before imaging), showing accumulation of ICG-labelled cells in and around all six laser lesions. (F,G) Corresponding fluorescein angiography images of the superficial retina and deep retina/choroid obtained 10 min after i.p. injection with 100 µl of fluorescein dye and imaged with a blue-light filter (488-nm solid-state excitation laser and 500-nm long-pass filter). (G) Deep retinal/choroidal images show that only two of six laser lesions subsequently developed choroidal neovascularization, and none of these 1-week-old lesions showed obvious fluorescein dye leakage into the surrounding tissues, suggesting that inflammatory cellular infiltration occurred independently of vascular leakage.

Fig. 5.

In vivo quantification of laser-induced CNV-related infiltration. (A) ICG was injected i.p. at day 0 before laser induction of CNV at day 3 in C57BL/6 mice. Imaging with the scanning laser ophthalmoscope using a near-infrared filter (790-nm diode excitation laser and 800-nm barrier filter) was performed on days 5, 7 and 10. (C-E) Increasing cellular infiltration in and surrounding the laser-CNV lesion was observed over time. (B,C′-E′) We demonstrate that the mean intensity values and standard deviations can be quantified over a given area and show an increase in inflammation over time. The mean intensity value of the lesion at day 5 is represented by the black histogram (C′), day 7 the superimposed light grey histogram (D′) and day 10 the superimposed medium grey histogram (E′).

Fig. 6.

Characterization of in vivo ICG-labelled cells in a laser-induced CNV murine model. The animal was injected i.p. with ICG 10 days before imaging and laser CNV induced 7 days before imaging. Fifty microlitres of a CD11b antibody conjugated to fluorescein isothiocyanate (FITC) was injected into the tail vein before being imaged at 15 and 30 min post-injection to assess the identity of ICG-labelled cells. A blue-light filter (488-nm solid-state excitation laser and 500-nm barrier filter) was used for the detection of cell labelling by CD11b-FITC. A scanning laser ophthalmoscope was used to acquire all images. (A) An infrared-reflectance (IR) image (820-diode excitation laser, no barrier filter) demonstrating the presence of laser-induced CNV lesions in the deep retina/choroid. (B) A deep retinal/choroidal image obtained with a near-infrared filter (790-nm diode excitation laser and 800-nm barrier filter), showing ICG-labelled cells surrounding two laser-induced CNV lesions. (C) A magnified view of ICG-labelled cells surrounding the CNV lesion from the top lesion in (B). (D,E) A deep retinal/choroidal image of the same mouse shown in (B) at 15 min (D) and 30 min (E) after a tail vein injection of CD11b-FITC. (F) A magnified view of CD11b-FITC-labelled cells surrounding the CNV lesion from (E), showing co-labelling of most but not all ICG-labelled cells.