|Molecular Vision 2006;
Received 13 September 2006 | Accepted 12 October 2006 | Published 26 October 2006
Intravital microscopy of leukocyte-endothelial dynamics using the Heidelberg confocal laser microscope in scleritis and allergic conjunctivitis
Lyndell L. Lim,1
Lani Hoang,1 Tiffany Wong,1
Stephen R. Planck,1,2,3
Mischa B. Ronick,1
Robert R. Gould,1
William D. Mathers,1
James T. Rosenbaum1,2,3
1Department of Ophthalmology, 2Department of Medicine, and 3Department of Cell and Developmental Biology, Casey Eye Institute, Oregon Health & Science University, Portland, OR
Correspondence to: Dr. Lyndell L. Lim, Centre for Eye Research Australia, University of Melbourne, 32 Gisborne Street, East Melbourne, Victoria 8002, Australia; Phone: +613-9929-8399; FAX: +613-9929-8379; email: email@example.com
Purpose: To examine leukocyte-endothelial cell rolling and arrest in human ocular vessels overlying sites of inflammation in various ocular inflammatory diseases in comparison to normal controls using the Heidelberg confocal laser microscope, which provides images with greater clarity and resolution than the tandem scanning microscope that uses white light.
Methods: Healthy controls (n=8) and patients with active anterior scleritis (n=7) or allergic eye disease (n=4) were scanned using the Heidelberg confocal laser microscope (HRT II) with the Rostock cornea module attachment for a minimum of 5 min at a depth of 45-120 μm from the conjunctival epithelial surface.
Results: There was a marked increase in the number of rolling leukocytes in scleritis patients (534+/-119 cells per mm2/min) versus controls (6+/-6 cells per mm2/min; p=0.0002) or allergic patients (59+/-44 cells per mm2/min; p=0.009). No statistically significant increase was seen in allergic patients compared to controls (p=0.059). A similar pattern was seen in the number of arrested leukocytes in patients with scleritis (56+/-23 cells per mm2) in comparison to either those with allergic eye disease or controls (each=0 cells per mm2; p=0.02).
Conclusions: Patients with scleritis have a significantly increased number of rolling and arrested leukocytes in superficial ocular vessels in comparison to patients with mild allergic conjunctivitis and controls. The image quality with this microscope is superior to prior studies with a scanning microscope.
Although leukocyte rolling and arrest are considered fundamental components of inflammation, almost all knowledge of this process in vivo derives from animal studies. The human eye is one of the few organs whose vessels are non-invasively accessible for imaging. To date, this has been limited to a tandem scanning microscope that uses white light [1-5].
Recent advances in confocal microscopes designed for use in patients have provided images of the conjunctival and episcleral vasculature with greater clarity and resolution (Figure 1). In this present study, we sought to examine leukocyte-endothelial cell rolling and sticking in ocular vessels overlying sites of inflammation using the Heidelberg confocal laser microscope in patients with scleritis, allergic conjunctivitis and normal controls.
Healthy controls and patients with active anterior scleritis or allergic eye disease provided informed consent and were prospectively enrolled from March, 2005 through March, 2006. OHSU Institutional Review Board permission was granted for the conduct of this study.
Prior to imaging, the surface of the eye was anesthetized with one drop of 0.5% proparacaine hydrochloride (Ophthetic; Allergan Optical, Irvine, CA) and lubricated with GenTeal Gel (Novartis Ophthalmics, East Hanover, NJ). All subjects were scanned using the Heidelberg Confocal Laser microscope (HRT II, Heidelberg Engineering, Heidelberg, Germany) with the Rostock Cornea Module attachment for a minimum of 5 min at a depth of 45-120 μm from the conjunctival epithelial surface. Images of vessels were captured for at least 20 s per field of view at a rate of 5 frames per s and a magnification of 400X. The ASL-1000 (Advanced Scanning Inc, New Orleans, LA) image shown in Figure 1 was captured as previously described .
In those patients with scleritis, vessels overlying areas of active scleritis were imaged, whereas in all other subjects, vessels 2 to 4 mm from the superior limbus were imaged. In one patient with active scleritis, images were taken before and 8 weeks after the institution of systemic immunosuppressive therapy. The average length and diameter of the vessels scanned were 434 μm and 55 μm, respectively.
Twenty clips of the raw movies were stabilized using MediaCybernetics Image Pro Plus Version 5 (Silver Spring, MD) to minimize the effects of eye movement during the recording. The images were then graded according to the number of arrested and rolling leukocytes per mm2 of vessel endothelium using a previously published method by Becker et al. . Two graders were used: one who was unmasked as to the diagnosis of the subject (Observer 1) and the other who was masked (Observer 2).
Statistical analysis consisted of an unpaired one-tailed t test based upon the hypothesis that there would be increased rates of leukocyte rolling and arrest in scleritis and allergic conjunctivitis subjects in comparison to controls. Prism 4 software (GraphPad Software, San Diego, CA) was used for all statistical calculations.
Nine controls, seven patients with scleritis, and four with mild allergic conjunctivitis were enrolled. Specific demographic data is shown in Table 1 and representative movies from each of these three subject groups are shown in Figure 2, Figure 3, and Figure 4.
As shown in Figure 5 and Figure 6, Observer 1 (unmasked) found a marked increase in the number of rolling leukocytes in scleritis patients (534±119 cells per mm2/min) versus controls (6±6 cells per mm2/min; p=0.0002) or allergic patients (59±44 cells per mm2/min; p=0.009). No statistically significant increase was seen in allergic patients compared to controls (p=0.059). A similar pattern was seen in the number of arrested leukocytes in patients with scleritis (56±23 cells per mm2) in comparison to either those with allergic eye disease or controls (each=0 cells per mm2; p=0.02).
Observer 2 (masked) had comparable findings to Observer 1 (Figure 5 and Figure 6), as this observer also found a marked increase in the number of rolling leukocytes in scleritis patients (570±121 cells per mm2/min) versus controls (154±65 cells per mm2/min; p=0.004). Again, no statistically significant increase was detected in allergic subjects compared to controls (47±47 cells per mm2/min; p=0.15). Additionally, a similar pattern was seen with arrested leukocytes in patients with scleritis (62±17 cells per mm2) in comparison to either the controls (6±6 cells per mm2; p=0.003) and those with allergic eye disease (0 cells per mm2; p=0.02). The only discrepancy seen between the two observers was in the rates of rolling leukocytes recorded in the control group that is most likely due to bias on the part of the unmasked observer.
In the one scleritis patient with pre and post treatment scans, the number of arrested cells prior to treatment (110 cells per mm2) had decreased to 18 cells per mm2 eight weeks after the commencement of treatment with oral prednisone (1 mg/kg) and methotrexate (20 mg/week).
Characteristics of rolling and sticking leukocytes in vessels of patients with various inflammatory diseases have been previously published [1-4], however they are yet to be described in scleritis. In addition, imaging of these leukocyte-endothelial dynamics has not been described in any condition with the Heidelberg confocal laser microscope (HRT II) and Rostock cornea module attachment, which affords far higher image quality in comparison to the standard, white light confocal microscopes that are used for human corneal imaging in vivo.
Our results have conclusively shown that leukocyte rolling and arrest are significantly increased in patients with scleritis in comparison to normal controls and those with mild allergic conjunctivitis with good inter-observer agreement. However, our results for allergic conjunctivitis were not as conclusive as previous studies . This discrepancy is most likely due to the patient population, as all of our patients had mild disease, with their diagnosis based upon the presence of mild symptoms of epiphora and pruritus. Examination of these patients revealed only mild conjunctival injection and no other signs of allergic conjunctivitis (such as papillary changes or follicles). In comparison, Helinto et al.  exposed the patients directly to an allergen to which the subjects were known to be sensitive, thereby precipitating a much more fulminant allergic inflammatory response. We also cannot exclude the possibility that study of a larger patient population would have demonstrated differences in rolling between allergic conjunctivitis patients and controls that were statistically significant.
Our rates of leukocyte rolling and sticking in scleritis subjects are also far greater than in previously published studies of allergic conjunctivitis and post surgical inflammation. Kirveskari et al. [1-4] described up to 78.8±40 cells/mm2/min rolling leukocytes in allergic patients and up to 53±34 cells/mm2/min in post surgical subjects. In comparison, our rates in scleritis patients are 10 fold greater. There are many possible explanations for this observation. The first is that the greater numbers of rolling and arrested leukocytes may be a reflection of the far greater degree of inflammation induced by scleritis in comparison to these other inflammatory conditions. This is supported by the reduction in the number of arrested leukocytes seen in the one patient who had pre and post treatment scans. The second possibility is that the improved image quality seen with the laser confocal microscope has resulted in a better ability to visualize and quantify the cells.
Ideally, we would like to correlate the degree of leukocyte rolling and sticking with the severity of the patient's scleritis, and possibly provide some prognostic information regarding the likely course of the patient's disease, based upon the change in leukocyte-endothelial dynamics with treatment. However, at this point, our patient set is too small and lacks sufficient sequential scans to draw any such comparisons or conclusions.
In conclusion, we have shown that laser confocal microscopy is a viable, noninvasive means of visualizing the human vascular bed relevant to several ocular inflammatory diseases and that the image quality with this microscope is superior to that obtained in prior studies with a scanning white light microscope. Patients with scleritis clearly have a significantly increased number of rolling and arrested leukocytes in superficial ocular vessels in comparison to patients with mild allergic conjunctivitis and controls. This demonstration of rolling and arrested leukocytes in scleritis with laser confocal microscopy holds future promise for its application in the assessment of disease severity, recognition of disease subsets, determining efficacy and mechanism of medications, and prediction of disease outcomes.
This study was supported by unrestricted funds to the Casey Eye Institute, J.T.R. and S.R.P. from Research to Prevent Blindness, New York, and the Stan and Madelle Rosenfeld Family Trust, Portland, OR.
1. Helinto M, Renkonen R, Tervo T, Vesaluoma M, Saaren-Seppala H, Haahtela T, Kirveskari J. Direct in vivo monitoring of acute allergic reactions in human conjunctiva. J Immunol 2004; 172:3235-42.
2. Kirveskari J, Vesaluoma MH, Moilanen JA, Tervo TM, Petroll MW, Linnolahti E, Renkonen R. A novel non-invasive, in vivo technique for the quantification of leukocyte rolling and extravasation at sites of inflammation in human patients. Nat Med 2001; 7:376-9.
3. Kirveskari J, Helinto M, Moilanen JA, Paavonen T, Tervo TM, Renkonen R. Hydrocortisone reduced in vivo, inflammation-induced slow rolling of leukocytes and their extravasation into human conjunctiva. Blood 2002; 100:2203-7.
4. Kirveskari J, Helinto M, Saaren-Seppala H, Renkonen R, Tervo T. Leukocyte rolling and extravasation in surgical inflammation after mechanical and laser-induced trauma in human patients. Exp Eye Res 2003; 77:387-90.
5. Nguyen TH, Dudek LT, Krisciunas TC, Matiaco P, Planck SR, Mathers WD, Rosenbaum JT. In vivo confocal microscopy: increased conjunctival or episcleral leukocyte adhesion in patients who wear contact lenses with lower oxygen permeability (Dk) values. Cornea 2004; 23:695-700.
6. Becker MD, Nobiling R, Planck SR, Rosenbaum JT. Digital video-imaging of leukocyte migration in the iris: intravital microscopy in a physiological model during the onset of endotoxin-induced uveitis. J Immunol Methods 2000; 240:23-37.