Confocal Intravital Microscopy of Leukocyte–Endothelial Dynamics Using the Heidelberg Confocal Laser Microscope in Patients With Scleritis and Allergic Conjunctivitis

Abstract

 

Purpose:

 

Imaging of leukocyte rolling and arrest in various inflammatory eye conditions is well established but has thus far been limited to a tandem scanning microscope that uses white light. Recent advances in the form of laser confocal microscopy have provided images with greater clarity and resolution (see 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.

 

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 minutes at a depth of 45–120µm from the conjunctival epithelial surface. In those with scleritis, vessels overlying areas of active scleritis were imaged, whereas in all other subjects, vessels 2–4mm away from the superior limbus were imaged

 

Results:

 

There was a marked increase in the number of rolling leukocytes in scleritis patients (1039+/–261 cells per mm²/min) vs. controls (15+/–15 cells per mm²/min, p=0.001) and allergic patients (145+/–112 cells per mm²/min, p=0.036). No statistically significant difference was seen between allergic patients and controls (p=0.127). A similar pattern was seen in the number of arrested leukocytes in patients with scleritis (50+/–52 cells per mm²) in comparison to either those with allergic eye disease or controls (each = 0 cells per mm², p=0.04). In the one scleritis patient with pre and post treatment scans, the number of arrested cells prior to treatment (126 cells per mm²) decreased post treatment (to 23 cells per mm²).

 

Conclusions:

 

Patients with scleritis have a significantly increased number of rolling and arrested leukocytes in superficial ocular vessels in comparison to patients with allergic conjunctivitis and controls. The image quality with this microscope is superior to prior studies with a scanning microscope.  

 

Keywords: microscopy: confocal/tunneling • inflammation • sclera