A novel non-invasive, in vivo technique for the quantification of leukocyte rolling and extravasation at sites of inflammation in human patients

Methods Patients. Ten otherwise healthy patients with senile cataract were enrolled for the study. Informed consent was obtained from all patients and the study protocol was reviewed by the Ethical Committee of the Helsinki University Eye and Ear Hospital according to the Declaration of Helsinki. Before selecting the patients the following exclusion criteria were applied: use of 1) anticoagulative, 2) immunosuppressive, and/or 3) anti-inflammatory therapies with the exception of a continuous prophylactic acetosalicylic acid (< 250 mg/day) treatment. All the patients were analyzed 7-15 d before the operation (PRE), 1 d after operation when the inflammation was most prominent (INF), and 24-42 d after the operation when the inflammation had resolved (POST). The blood leukocyte count and their subclasses were analyzed from PRE and INF patients (Table 1). The cataract operation was performed according to standard phacoemulsification technique in subtenon's anesthesia. The surgery began by making a temporal conjuctival incision and cauterizing bleeding episcleral vessels. The wounds were self-sealing with no stitches and the conjunctiva was apposed to the wound by cautery. All patients received topical ofloxacin 3 mg/ml (Exocin, Allergan, Irvine, California) 1-2 drops × 4 for 3 preoperative days and dexametasone 1 mg/ml and chloramphenicol 2 mg/ml (Oftan DexaChlora, Santen, Tampere, Finland) 1-2 drops × 4 for 3 postoperative weeks. In vivo confocal microscopy. A tandem scanning confocal microscope was used (TSCM, Model 165A, Tandem Scanning Corporation, Reston, Virginia) equipped with a X24/0.6 NA immersion objective lens. Before the examination one drop of topical anesthetic (benoxinate hydrochloride, Oftan Obucain, Santen, Tampere, Finland) was applied on the ocular surface and one drop of 2.5% hydroxymethylcellulose gel (Goniosol, IOLAB Pharmaceuticals, Claremont, California) on the objective tip. The patient was fixed with the contralateral eye on a bright object to minimize eye movements during examination, which lasted 10-15 min for each eye. The objective lens of the microscope was adjusted to give an en face view of the bulbar conjunctival vessels about 3-6 mm lateral to the limbus. The setup and operation of the confocal microscope have been described. Briefly, the illumination was supplied by a 100-W mercury lamp. The internal lenses of the objective were moved with a motorized focusing device (Oriel 18011 Encoder Mike TM Controller, Stratford, Connecticut) interfaced to a personal computer to vary the focal plane relative to the objective tip. Real-time images were captured using a low-light video camera (VE-1000 Sit System, Dage-MTI, Michigan City, Indiana). The black level, gain and kV were automatically controlled. With this objective and camera, the field-of-view was 450 × 360 μm, and the optical slice thickness (z-axis resolution) was 9 μm. From the camera, the video stream was directly captured by a video capturing card (Matrox Marvel G-200, Matrox Electronic Systems, Quebec, Canada) in a second computer by Matrox PC-VCR software and saved on-line to the hard disk in Windows AVI format (continuous data flow 2 Mbytes/s, 25 frames/s, PAL standard). The primary files were roughly edited and split off-line by Quick Time 4.0 Pro (Apple Computer, Austin, Texas) and saved in CD-R disks. The final video analysis was performed using Adobe Premiere 5.1 (Adobe Systems, San Jose, California), and image analysis and processing was performed with Adobe Photoshop 5.0, Scion Image (NIH Image, Scion, Frederick, Maryland) and Corel Draw 9 (Corel, Ottawa, Ontario, Canada) software. Leukocyte-rolling and histological image parameters. Vessel diameters and the number of rolling cells were measured from all vessels. Mean centerline flow velocity was counted from 3-5 freely moving bright cells by measuring average movement in 4 subsequent frames. An estimate of shear rate was calculated as follows: WSR = 8 × V_leuk/D, where V_leuk is the leukocyte velocity in the centerline of the vessel and D is the vessel diameter. The number of rolling cells was counted from each vessel with continuous flow and sharp image from the cells passing an imaginary horizontal line in the vessel, which was fixed in one of the local landmarks in the vessel area to eliminate the effect of small movements. The velocity was measured from 28% of 170 rolling cells. The number of extravasated leukocytes in a focal plane of conjunctival stroma was counted from an area of 0.1 mm² adjacent to the vessels where rolling had been analyzed. Statistics. Wilcoxon matched-pairs signed-ranks sum test was used to compare the different groups (PRE, INF, POST) against each parameter. P-values were adjusted with the Bonferroni method and values of less than 0.05 were considered significant.