Confocal microscopy reveals persisting stromal changes after myopic photorefractive keratectomy in zero haze corneas

Abstract

Aims: Micromorphological examination of the central cornea in myopic patients 8-43 months after excimer laser photorefractive keratectomy (PRK), using the slit scanning confocal microscope.

Methods: Patients were selected from a larger cohort of individuals on the basis of full corneal clarity (haze grading 0 to +1; mean 0.3) and their willingness to participate in the study. 15 eyes of 10 patients with myopic PRK (-4 to -11 D; mean 6.7) and an uneventful postoperative interval of 8-43 months (mean 26) were examined. Contact lenses had been worn by eight of the 10 patients for 4-11 years (mean 6.7) before surgery. Controls included the five untreated fellow eyes of PRK patients, 10 healthy, age matched volunteers without a history of ocular inflammation or contact lens wear, and 20 patients who had worn rigid gas permeable (n = 10) or soft contact lenses (n = 10) for 2-11 years. Subjects were examined with a real time flying slit, scanning confocal microscope using x25 and x50 objectives.

Results: In PRK treated patients and contact lens wearers, basal layer epithelial cells sporadically displayed enhanced reflectivity. The subepithelial nerve plexus was observed in all individuals, but was usually less well contrasted in the PRK group, owing to the presence of a very discrete layer of subepithelial scar tissue, which patchily enhanced background reflectivity. Within all layers of the stroma, two distinct types of abnormal reflective bodies were observed in all PRK treated eyes, but in none of the controls. One had the appearance of long (> = 50 microns), slender (2-8 microns in diameter) dimly reflective rods, which sometimes contained bright, punctate, crystal-like inclusions, arranged linearly and at irregular intervals. The other was shorter (< 25 microns), more slender in form (< 1 micron in diameter), and highly reflective; these so called needles were composed of crystal-like granules in linear array, with an individual appearance similar to the bright punctate inclusions seen in rods, but densely packed. Both of these unusual structures were confined, laterally, to the ablated area, but were otherwise distributed throughout all stromal layers, with a clear predominance in the anterior ones. These rods and needles were observed in all PRK treated corneas, irrespective of previous contact lens wear. On the basis of qualitative inspection, the incidence of rods and needles did not appear to correlate with either the volume of tissue ablated or the length of the postoperative interval. In contact lens wearing controls, highly reflective granules, reminiscent of those from which the needles were composed, were found scattered as isolated entities throughout the entire depth and lateral extent of the corneal stroma, but rods and needles were never encountered. The corneal endothelium exhibited no obvious abnormalities.

Conclusion: Confocal microscopy 8-43 months after PRK revealed belated changes in the corneal stroma. These were manifested as two distinct types of abnormal reflective bodies, which had persisted beyond the stage when acute wound healing would have been expected to be complete. The clinical significance of these findings in the context of contrast visual acuity and long term status of the cornea is, as yet, unknown.

Figure 1

Basal layer of corneal epithelium. Top: normal control eye, depicting non-reflective cells delimited by dimly reflective borders. Bottom: PRK patient 1 year after surgery, illustrating the scattered occurrence of individual cells with increased reflectivity (arrows), a phenomenon which may be indicative of mitotic cell division. ×50 objective; bar= 50 µm.

Figure 2

Subepithelial nerve plexus. Top: normal control eye, depicting beaded nerve fibres enhanced by an optically unstructured, and hence non-reflective, Bowman's layer. Bottom: PRK patient 1 year after surgery. This subepithelial nerve fibre (arrow) is less well contrasted than those in controls owing to the presence of a discrete layer of scar tissue, which increases background reflectivity. ×50 objective; bar=50 µm.

Figure 3

Keratocyte nuclei in the most anterior stromal layer. Top: normal control eye. Keratocyte nuclei manifest a characteristic polygonal shape; they are regularly spaced. Bottom: PRK patient 2 years after surgery. The most anteriorly located keratocytes have assumed this pseudoanterior position owing to ablation of the superficial stroma. Cell nuclei are partially masked by the subepithelial scar tissue which increases background reflectivity. Dimly reflective rods (arrow), approximately 2-8 µm in diameter and >50 µm in length are visible (large arrow), some of which contain highly reflective punctate inclusions (small arrows). ×50 objective; bar=50 µm.

Figure 4

Mid stroma (about 250 µm below the basal epithelial cell layer). Top: control cornea from a patient with an 8 year history of soft contact lens wear. Note the presence of scattered highly reflective spots (arrows) less than 1 µm in diameter. Bottom: PRK patient with a 7 year history of soft contact lens wear, 1 year after surgery. The distribution pattern of keratocyte nuclei appears normal. This micrograph illustrates dimly reflective rods (small arrow) with brilliant, punctate inclusions, and a group of highly reflective needles composed of linearly arranged, densely packed granules (large arrow). ×50 objective; bar=50 µm.

Figure 5

Anterior to mid stroma. Top: anterior to mid stroma (about 150 µm beneath the basal epithelial cell layer), 2 years after PRK. The broad, long, and tapering body running diagonally across the centre of the picture (large arrow) is probably a cell process classified as a dimly reflective rod, which contains highly reflective granules (small arrows). Bottom: mid stroma (about 200 µm beneath the basal epithelial cell layer), 1 year after PRK, illustrating rods (arrowhead) and needles (arrow) oriented predominantly parallel to the corneal surface. ×50 objective; bar=50 µm.

Figure 6

Posterior stroma (less than 50 µm above Descemet's membrane). Top: normal control eye, illustrating the oval or rhomboid form of nuclei characteristic of keratocytes within this layer. Bottom: PRK patient 2 years after surgery, illustrating dimly reflective rods some with bright, punctate inclusions (arrowheads), and a needle (large arrow). Some punctate inclusions in association with a keratocyte nucleus are also visible (small arrows). ×50 objective; bar=50 µm.