Bulbar conjunctival microvascular responses in dry eye

Abstract

Purpose: Conjunctival microvascular responses may be a surrogate metric of efferent neural pathway function innervating the ocular surface as changes in blood flow occur within seconds after a stimulus. As somatosensory dysfunction may partially underlie dry eye (DE), in this study we evaluate whether bulbar conjunctival microvascular alterations correlate with various aspects of DE.

Methods: Fifty-six DE patients were prospectively recruited from a Veterans Affairs ophthalmology clinic over an 11-month period. DE symptoms and ocular pain were assessed along with DE signs. A novel functional slit lamp biomicroscope (FSLB) was used to image the temporal bulbar conjunctiva from the right eye before and after central corneal stimulation with an air puff. Blood flow velocities were measured and noninvasive microvascular perfusion maps (nMPMs) were created.

Results: The bulbar blood flow velocity was 0.50 ± 0.15 mm/s at baseline and increased to 0.55 ± 0.17 mm/s after stimulation (P < 0.001); the average change in velocity was 0.05 ± 0.09. nMPMs values and venule diameter, on the other hand, did not significantly increase after stimulation (1.64 ± 0.004 at baseline, 1.65 ± 0.04 after stimulation, P = 0.22 and 22.13 ± 1.84 μm at baseline, 22.21 ± 2.04 μm after stimulation, P = 0.73, respectively). Baseline blood flow velocity positively associated with Schirmer scores (r = 0.40, P = 0.002). Those with higher self-rated wind hyperalgesia demonstrated less change in blood flow velocity (r = -0.268, P = 0.046) after air stimulation on the central cornea.

Conclusion: Conjunctival blood flow velocity, but not vessel diameter or complexity, increases after wind stimuli. Baseline flow positively correlated with Schirmer scores while change in flow negatively correlated with self-reported wind hyperalgesia.

Keywords: Blood flow velocity; Corneal sensitivity; Fractal dimension; Functional slit lamp biomicroscopy; Neuropathic ocular pain.

Figure 1

Temporal bulbar conjunctiva microvasculature imaged by the functional slit lamp biomicroscope. Videos clips were acquired from six locations which were homogeneously located on the temporal side of the bulbar conjunctiva (A). With ×175 magnification, red blood cell clusters (white arrow) are shown in the six fields (a–f).

Figure 2

Measurement of the vessel diameter and blood flow velocity. Custom software was developed and utilized to process the video clips obtained on the bulbar conjunctiva. The first frame (A) of the video clip was used for registering all frames to compensate for the eye motion. After that, all registered images were averaged and the blood vessels were segmented from the average image. The vessels were automatically identified and marked in numbers (B). The vessel walls were outlined and marked in green and blue lines for measuring the vessel diameter (B). By calculating the image intensity within the areas defined by the vessel walls, an intensity profile along the center line between these walls was generated for each frame. Using all intensity profiles of all frames in the video clips, a space-time image was obtained and used to measure the blood flow velocity. The slopes of the bands (i.e. moving distance over time) were manually outlined (marked in red lines) and calculated as the measurements of axial blood flow velocity (C for vessel No. 8, D for vessel No. 19).

Figure 3

Non-invasive microvascular perfusion maps (nMPMs) and fractal analysis. Custom software was developed to segment conjunctival vessels and to create the nMPMs for fractal analysis using a series of image processing procedures. The raw image was first resized from 3,456 × 2,304 pixels to 1,024 × 683 pixels (A). Imaging processing using morphological opening was performed to create nMPMs (B). Vessels were segmented (C) and the image was cropped a field of view 7.85 × 7.85 mm² containing 512 × 512 pixels (D). The cropped image was then inverted (E) and skeletonized (F). The image was further inverted back for fractal analysis (G) using monofractal analysis (i.e. box-counting, H). Bars=3 mm.

Figure 4

Bulbar conjunctival blood flow velocities, nMPMs, and vessel diameters were measured in DE patients before and after air stimulation. A. Blood flow velocity increased significantly after air stimuli (P<0.001). B, C. Both fractal dimension and vessel diameter were not significantly different before and after air stimulation (P=0.22, P=0.73, respectively).

Figure 5

An inverse correlation is noted between change in conjunctival microvascular blood flow after mechanical stimulation and self-reported wind hyperalgesia (r=−0.268, P=0.046).