Video Report July 2001
Evaluation of leukocyte dynamics in mouse retinal circulation with scanning laser ophthalmoloscopy
Heping Xu a, A. Manivannan b, Garry Daniels, Janet Liversidge a, Peter F. Sharp b, John V. Forrester a, Isabel J. Crane a
a Department of Ophthalmology; b Department of Biomedical Physics and
Bioengineering, Aberdeen University Medical School, Scotland, United Kingdom
Correspondence to: Dr. Isabel Crane, Department of Ophthalmology, Aberdeen University Medical School, Foresterhill, Aberdeen
AB25 2ZD, Scotland, United Kingdom
Accepted for publication 15 May 2001
The video shows fluorescently labelled leukocytes moving in the retinal circulation of B10 RIII mice. Normal (part 1), EAU, 9 days post-immunization (part 2), and EAU 16 days post-immunization (part 3). Leukocytes move more slowly and roll in the veins and venules of diseased mice. At day 16 the are many new blood vessels and less rolling. In part 4 the retinal and less clearly choroidal circulation is seen in a normal BALB/c mouse. View Video
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Leukocyte-endothelial cell interactions play an important role in the pathogenesis of various types of retinal vascular diseases, including diabetes, uveitis, and ischemic lesions. Over the last few years, several methods have been devised in which the scanning laser ophthalmoscope (SLO) is used to study leukocyte-endothelial interactions in vivo [1,2]. Previously we reported a noninvasive in vivo leukocyte tracking method using the SLO in rat. In this method, a nontoxic fluorescent agent (6-carboxyfluorescein diacetate, CFDA) was used to label leukocytes in vitro. Leukocyte velocities within the retinal and choroidal circulations were be quantified simultaneously . None of the previous methods has been developed for imaging the murine fundus, mainly due to problems arising from the small size of the mouse eye. However, there are many advantages of using a murine model to study retinal vascular diseases such as enhanced genetic definition, increased range of reagents available for immunological studies and cost reduction. We have developed our SLO method such that we can track leukocytes in the mouse retinal and choroidal circulations.
Experimental autoimmune uveitis (EAU) was induced in B10 RIII mice by subcutaneous injection of human IRBP peptide 160-180, 50mg in 100ml emulsified 1:1 CFA. Mice, 8-12 weeks old (normal BALB/c mice, normal B10 RIII mice and EAU induced B10 RIII mice at day 9 and 16 post-immunization, (p.i.)) were anesthetized with an intramuscular injection of 0.4 ml/kg hypnorm and intraperitoneal administration of 1 ml/kg diazepam, producing deep anaesthesia for 45 minutes. Pupils were dilated with 0.5% cyclopentolate. Hard contact lenses (radius of curvature, 1.7mm; diameter, 3.2mm; Cantor & Nissel, UK) were placed on the mouse cornea to obtain a clear view of the fundus. Animals were kept immobile and manoeuvred for examination using a perspex restraint mounted on a tripod. 2x107 leukocytes from syngeneic mouse spleens were incubated with 40mg/ml calcein-AM (C-AM, Molecular Probes Europe BV, Leiden, The Netherlands) at 37°C for 30 minutes. Mouse retinal images were examined using an SLO. An argon laser (wavelength 488nm, power = 1mW) was used to excite fluorescence and a 520nm barrier filter was used to detect fluorescing cells. A low dose of 100ml 0.05% sodium fluorescein was injected via the tail vein to outline the retinal and choroidal vasculature followed by 1x107 C-AM labeled leukocytes in 150ml RPMI 1640. Images were recorded simultaneously on videotape (S-VHS) and digitally at 25 frames per second with a resolution of 768x576 pixels, using a frame grabber (Meteor, Matrox, Swindon, UK) linked to a PC. In each animal three fundus areas adjacent to the optic disc were chosen and the animal was positioned so that a retinal artery and vein could be viewed in each area. The laser focus was adjusted to visualize different layers of the fundus.
Our method of measuring leukocyte hemodynamics in the retina has the advantages that leukocyte labeling with calcein-AM is nontoxic and has no effect on cell adhesion. In addition, only one image frame is required to determine leukocyte velocity  making measurement easier and more accurate than other methods. Improved computer software enables us to measure the exact distance moved by the cell through a tortuous vessel. Leukocyte dynamics can thus be studied in small vessels which are areas of interest for most microvascular disease.
A higher resolution image and higher magnification of SLO are used to visualize the smaller vessels of the mouse retina. Two other technical problems have been resolved. Firstly, unlike the human lens, the mouse lens is spheroid and after pupil dilation, the retinal image is distorted due to the spherical aberration. To avoid this distortion, an extra lens (+25D) is placed 1 cm in front of the mouse eye to further focus the laser beam. This ensures that most of the laser beam passes through the centre of the mouse lens, and spherical aberration is reduced. Secondly, the retention of the tear film during the experiment is very important. Once the tear film has evaporated, the mouse cornea becomes dry and cloudy. The use of a contact lens effectively solved this problem.
In nonpigmented mice (BALB/c mice), leukocyte behavior can be observed in both choroidal and retinal circulations. However, because of the strong choroidal background fluorescence during angiography, retinal capillaries can not be clearly observed. In contrast, in pigmented mice (B10 RIII mice), retinal microcirculation is clear against the dark background of the choroid, while choroidal circulation is masked behind the pigment epithelial layer and can not be visualized at all. In B10 RIII mice at peak level of clinical EAU the cloudiness of the optical media and the extravasation of fluorescein blurs retinal images. However, leukocyte behavior is clearly visible until day 9 and at day 16 p.i. At day 9 p.i. retinal veins are dilated and there is fluorescein dye leakage, rolling cells can be seen in retinal veins and venules (rolling efficiency 9.39�1.49 %). At day 16 p.i. clinical disease was still apparent and SLO showed that the retinal vessels were abnormal. However less cells rolled inside the vessels (rolling efficiency 5.83�0.92%, P<0.05 compared to day 9 p.i.).
To our knowledge, this study is the first to visualize and measure leukocyte dynamics in the murine retinal and choroidal microcirculations. This noninvasive method is of value for the study of retinal hemodynamics in murine models of ocular disease including EAU, diabetic retinopathy and retinal ischaemia.
We thank Mr. Hattam Atta and Ms Karon Robinson for their help with the design of the mouse contact lens.
This work was supported by The Wellcome Trust, grant No. 057311
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