Improved leukocyte tracking in mouse retinal and choroidal circulation.

The purpose of this study is to develop a new method with which to visualize leukocyte dynamics in murine choroidal and retinal circulation. Both pigmented (B10.RIII) and non-pigmented (BALB/c) mice were used in this study. One hundred microl of 0.05% sodium fluorescein was injected via the mice tail vein to outline the vessel, followed by 150 microl (10(7) cells) C-AM labelled leukocytes. Fundus images were obtained with a confocal scanning laser ophthalmoscope. The dynamic image sequences were recorded simultaneously on videotape (S-VHS) and digitally at 25 frames per sec. The digital images were later analysed with a custom-made personal computer-based image analysis system. Both the choroidal and retinal circulation can be visualized in non-pigmented mice in the first few seconds of fluorescein angiography. However, the view of the choroidal and the retinal capillary circulation is soon blurred due to the rapid fluorescein leakage in the choroid. In contrast, in pigmented mice, retinal circulation is clear against the dark background of the choroid, while choroidal circulation is masked behind the pigment epithelial layer and cannot be seen at all. C-AM labelled leukocytes were clearly seen in the retinal circulation of all experimental mice and in the choroidal circulation of non-pigmented mice for as long as 30 min. The number of labelled circulating cells decreased as time clasped. Cells moved rapidly in the retinal arteries, slowing down or even stopping for a few seconds in the capillary system, and then moved slightly faster again through the postcapillary venules and veins. In non-pigmented mice, significant number of cells were seen to have arrested in the choroidal circulation. There was no difference between B10.RIII mice and BALB/c mice in vessel diameters, leukocyte velocities and shear stresses. This method allows the visualization of leukocytes and provides data on their behavior as they move through the choroidal and retinal circulation of non-pigmented mice, and in the retinal circulation of pigmented mice. It provides a valuable new tool for the investigation of real time leukocyte dynamics in murine retinal and choroidal microcirculations both under physiological conditions and during the development of ocular disease.

[1]  D. Hatchell,et al.  Neutrophils plug capillaries in acute experimental retinal ischemia. , 1994, Microvascular research.

[2]  M. Shimura,et al.  Quantifying leukocyte dynamics and plugging in retinal microcirculation of streptozotosin-induced diabetic rats. , 1999, Current eye research.

[3]  S Chien,et al.  In vivo measurements of "apparent viscosity" and microvessel hematocrit in the mesentery of the cat. , 1980, Microvascular research.

[4]  M. Stanford,et al.  Leukocyte trafficking in experimental autoimmune uveitis in vivo , 1998, Journal of leukocyte biology.

[5]  B. Khoobehi,et al.  Study of lymphocyte dynamics in the ocular circulation: technique of labeling cells. , 1995, Current Eye Research.

[6]  Y. Ogura,et al.  Retinal leukocyte behavior in experimental autoimmune uveoretinitis of rats. , 1997, Experimental eye research.

[7]  T. Chan‐Ling.  Glial, vascular, and neuronal cytogenesis in whole‐mounted cat retina , 1997, Microscopy research and technique.

[8]  G. Schmid-Schönbein,et al.  Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy. , 1991, The American journal of pathology.

[9]  Y Yang,et al.  Measurement of retinal blood flow with fluorescein leucocyte angiography using a scanning laser ophthalmoscope in rabbits. , 1996, The British journal of ophthalmology.

[10]  P F Sharp,et al.  In vivo cell tracking by scanning laser ophthalmoscopy: quantification of leukocyte kinetics. , 1998, Investigative ophthalmology & visual science.

[11]  Y. Ogura,et al.  A new fluorescent imaging procedure in vivo for evaluation of the retinal microcirculation in rats. , 1995, Current eye research.

[12]  M. Paques,et al.  Evaluation of fluorescein-labeled autologous leukocytes for examination of retinal circulation in humans , 2000 .

[13]  S. D. House,et al.  Leukocyte-endothelium adhesion: microhemodynamics in mesentery of the cat. , 1987, Microvascular research.

[14]  K Miyamoto,et al.  In vivo demonstration of increased leukocyte entrapment in retinal microcirculation of diabetic rats. , 1998, Investigative ophthalmology & visual science.

[15]  Y. Ogura,et al.  In vivo quantification of leukocyte behavior in the retina during endotoxin-induced uveitis. , 1996, Investigative ophthalmology & visual science.

[16]  P F Sharp,et al.  Digital fundus imaging using a scanning laser ophthalmoscope. , 1993, Physiological measurement.

[17]  G. Nash,et al.  Effects of fluorescent dyes on selectin and integrin-mediated stages of adhesion and migration of flowing leukocytes. , 2000, Journal of immunological methods.

[18]  Y. Ogura,et al.  Quantitative evaluation of leukocyte dynamics in retinal microcirculation. , 1995, Investigative ophthalmology & visual science.