Confocal microscopy examines excimer wound healing

In vivo confocal microscopy may give clues to what causes good, bad refractive results.

Photorefractive keratectomy (PRK) and LASIK are the two primary procedures for correcting refractive errors using the excimer laser. In PRK, the epithelium is re moved, the exposed stroma is photoablated, and the epithelial defect heals in 2 to 4 days. The LASIK procedure re quires that a hinged flap (consisting of the surface epithelium, Bowman’s membrane and anterior stroma) be created using a microkeratome. The flap is folded back, the exposed stroma is photoablated using an excimer laser, and the flap is then replaced, covering the treated area. After either procedure, flattening of the central corneal curvature from tissue removal results in a decrease in refractive power in myopic correction.

Despite high success rates and increasing popularity worldwide, studies on PRK and LASIK have shown unexpected visual regression. There have been many studies on clinical outcomes after PRK and LASIK, but few of these reports address the biological changes associated with these surgeries. Recently, in vivo confocal microscopy has been introduced as a tool for the evaluation of wound healing at the cellular level in humans following refractive surgery. This procedure offers tremendous promise for improving surgical results.

Confocal microscopy advantages

A confocal microscope is a type of microscope in which a thick object, such as the cornea, is illuminated with a focused spot of light.

Because a conventional microscope collects all of the light reflected back through the objects that are being imaged, out-of-focus light that is above and below areas of interest in the focal plane will create unsatisfactory images. This poor imaging occurs in all but the thinnest specimens at high magnification. Furthermore, the need to section the sample mechanically, then fix, embed or stain specimens introduces artifacts that are uncontainable.

Confocal microscopy is a new imaging paradigm that overcomes these problems. The optical sectioning ability of confocal microscopy allows images to be obtained from different depths within thick tissue specimens, thereby eliminating the processing and sectioning procedures required with conventional light microscopy. Thus confocal microscopy has made it possible to view biological tissues under more physiologic conditions than were previously possible. The most widespread biological application of confocal microscopy has been in the localization of immunofluorescent lab eled proteins in cell cultures or within excised blocks of tissue.

Because of this noninvasive optical sectioning capability, confocal micro scopy is also ideal for in vivo systems for ocular studies, especially in the cornea. H. Dwight Cavanagh, MD, PhD, first applied the tandem scanning confocal microscope to the living human eye in 1989. Since that time, improvements have been made for higher quality imaging. Confocal microscopy through focusing technology has also been developed for measurement of corneal sub-layer thickness and estimation of the intensity of postoperative haze. Confocal microscopy through focusing technology is performed as a continuous z-axis scan through the entire cornea, from the front of the epithelium to below the endothelium.

Confocal microscopy after PRK

Corneal haze is one of the major complications associated with PRK. It may cause a decreased level of best corrected visual acuity. Although the mechanism of corneal haze is still un- identified, recent studies suggest that stromal wound healing and activation of keratocytes contribute to this process.

My colleagues and I performed a study using in vivo confocal micro scopy, showing normal quiescent keratocytes with low-reflecting nuclei in preoperative corneas and activated keratocytes in photoablated anterior stromal surface at 3 weeks after PRK. The reflectivity and density of keratocytes increased slightly at 6 weeks and peaked at 3 months with increased reflectivity surrounding dark cavity-like structures. At 6 months after PRK, moderately reflective keratocytes with decreased cell density were observed.

After PRK, stromal regrowth oc curred gradually in most patients who also had a normal wound-healing morphology and keratocyte density. This suggests that deposition of new extracellular matrix components is the main repair mechanism by which the stroma gradually regains thickness and curvature after PRK.

By contrast, the recurrence of epithelial thickening had no significant effect on refractive regression. Also, recurrence of stromal thickening was not as sociated with haze development over time measured by confocal microscopy, suggesting that haze and regression were caused by an independent wound-healing mechanism.

Several methods exist for epithelium removal prior to PRK, and many studies have been performed to identify the re moval technique that induces the least amount of post-PRK corneal haze and regression.

The study design was prospective and randomized, using in vivo confocal microscopy to compare the wound-healing process after PRK with mechanical epithelial debridement to the laser-scrape technique. Twenty eyes of 10 patients, with a mean spherical equivalent of –4.75 D ±0.94 D, were enrolled in this study. Prior to laser stromal ablation, one of two epithelial debridement methods was randomly assigned to the first eye and the other was assigned to the contralateral eye. For eyes receiving the laser-scrape technique, a standardized, central, 6-mm diameter laser photoablation performed in PTK mode with a 45-µm depth setting was used. This depth was chosen in order to prevent possible ablation of the basal lamina, as the average epithelial thickness was 51 µm ±4 µm as measured in a previous study.

The remaining thin layer of epithelium was gently removed with a blunt spatula. In preoperative corneas, epithelial thickness varied from 42.85 µm to 55.42 µm, averaging a thickness of 50.08 µm ±3.70 µm. The epithelium was significantly thinner at 3 weeks post-PRK, returning to the preoperative measurement by 6 months. The change of epithelial thickness values for different time periods after mechanical debridement appeared significantly different (P =.003). This finding suggests that the mechanical epithelial debridement may induce some epithelial hyperplasia over the 6-month period after PRK.

Alternatively, the changes in epithelial thickness may result from stromal surface irregularities because the epithelium remodels itself in compensation for induced stromal surface abnormalities. Stromal thickness values also showed no statistically significant difference be tween the two methods at any observational period. After PRK, 1 cornea showed grade 2 haze with slit-lamp biomicroscopy and 4 corneas showed grade 1 haze. The remaining eyes showed no haze after 1 month. However, confocal microscopic measurement of corneal haze showed significant increase of haze between baseline and post-PRK corneal haze in all patients, suggesting that confocal microscopy through focusing technology is more sensitive in measuring haze than slit-lamp examination.

There was no significant difference in both groups for confocal microscopy through-focusing measured haze. The two epithelial removal methods were compared in the same patients, excluding individual variability in the wound-healing process. Because of this, it is possible to conclude that there is little to no difference in haze after PRK between the epithelial removal techniques. This may also be true for other parameter studies (that is, epithelial and stromal thickness or depth of photoablation).

Confocal microscopy after LASIK

The tremendous increase in popularity of LASIK is due to good clinical results for myopia with minimal pain and a short visual recovery time. LASIK is performed worldwide with increasing frequency, despite the absence of complete data on the healing response and long-term complications at the tissue level. Several in vivo confocal microscopy studies were recently performed to evaluate wound-healing response after LASIK. These studies demonstrated that keratocyte activation induced by LASIK is of short duration compared with that of PRK.

One of the advantages of confocal microscopy through-focusing technology is precise measurement of flap thickness after LASIK. Because reproducible flap thickness is an important factor in LASIK surgery, the intention was to measure the flap thickness from two different microkeratomes and compare the results relative to intended thickness.

The intended measurements were a 160-µm flap thickness and 8.5-mm flap diameter for the Automated Corneal Shaper (ACS, Bausch & Lomb Surgical) and a 180-µm flap thickness and 8.5-µm flap diameter for the Hansatome (Bausch & Lomb Surgical).

The results for the ACS were an average flap thickness of 132.74 µm ±12.53 µm (110.73 µm to 145.55 µm). The Hansatome achieved an average flap thickness of 167.46 µm ±21.39 µm (142.94 µm to 209.40 µm). The ACS made thinner flaps in all patients than were intended, by an average of 27.26 µm ±12.53 µm. By contrast, 60% of pa tients treated with the Hansatome had thinner flaps than intended, by an average of 27.83 µm ±8.12 µm, and 40% of patients had thicker flaps than intended by an average of 10.39 µm ±11.12 µm. An increase in corneal epithelial thickness was also observed at 3 to 6 months after LASIK. In this study, the Han satome was found to cut a more precise flap than the ACS microkeratome, which cut thinner flaps than were predicted.

Minna H. Vesaluoma, MD, reported that confocal microscopy revealed microfolds and interface particles in almost every eye. These findings are not easily detected by slit-lamp biomicro scopy. The apparent loss of cells in the most anterior keratocyte layer began at 6 months after surgery. The reason for this may be suggested by loss of direct innervation of keratocytes by stromal nerve fiber or disruption of communication with more posterior keratocytes and the keratocytes surrounding the flap. These findings may be important factors for LASIK patients and should be confirmed by future long-term prospective studies.

In order to achieve better surgical success rates and lower rates of complications, more studies must be performed on wound-healing time in LASIK. Confocal microscopy provides a noninvasive method to assess corneal wound healing at the cellular level in human eyes. It may be studied sequentially, so that the amount and location of scarring and tissue reaction can be quantified. Assessing wound healing at the cellular level will also make the evaluation of the long-term effects of PRK and LASIK in individual patients possible and may lead directly to an understanding of why the laser-induced optical correction regresses in some patients and not in others. With further development and application, the in vivo confocal microscope has the potential to be a crucial tool in refractive surgery.

A note from the editors:

This article originally appeared in Vision Restoration: for achieving best uncorrected vision, a publication of SLACK Incorporated that is presented as a professional service by Allergan Surgical.

For Your Information:

• Young Ghee Lee, MD, PhD, is an assistant professor of ophthalmology at the Yonsei University College of Medicine in Seoul, Korea.

References:

Cavanagh HD, Jester JV, Essepian J, et al. Confocal microscopy of the living eye. CLAO J. 1990;16:65-73.

Li HF, Petroll WM, Moller-Pedersen T, et al. Epithelial and corneal thickness measurement by in vivo confocal microscopy through focusing. Curr Eye Res. 1997;16:214-221.

Moller-Pedersen T, Vogel M, Li HF, et al. Quantification of stromal thinning, epithelial thickness, and corneal haze after photorefractive keratectomy using in vivo confocal microscopy. Ophthalmology. 1997;104:360-368.

Lee YG, Chen WYW, Petroll WM, et al. Corneal haze following photorefractive keratectomy (PRK) using different epithelial removal techniques: Mechanical debridement versus laserscrape. Ophthalmology. 2000: [In press.]

Moller-Pedersen T, Cavanagh HD, Petroll WM, Jester JV. Stromal wound healing explains refractive instability and haze development after photorefractive keratectomy: A 1-year confocal microscopic study. Ophthalmology. 2000;107:1235-1245.

Reinstein DZ, Silverman RH, Sutton HFS, et al. Very high-frequency ultrasound corneal analysis identifies anatomic correlates of optical complications of lamellar refractive surgery. Ophthalmology. 1999;106:474-482.

Vesaluoma M, Petroll WM, et al. Corneal stromal changes induced by myopic LASIK. Invest Ophthalmol Vis Sci. 2000;41:369-376.

Latvala T, Barraquer-Coll C, Tervo K, Tervo T. Corneal wound healing and nerve morphology after excimer laser in situ keratomileusis (LASIK) in human eyes. J Refract Surg. 1996;12:677-683.

Slowik C, Somodi S, Richter A, Guthoff R. Assessment of corneal alterations following laser in situ keratomileusis by confocal slit scanning microscopy. Ger J Ophthalmol. 1997;5:526-531.