Ординатура / Офтальмология / Английские материалы / Wavefront Customized Visual Correction The Quest for Super Vision II_Krueger, Applegate, MacRae_2003

Figure 24-1. Eye movement coordinates and their generation from extraocular muscles.

ment coordinates are normally described in an eye-fixed coordinate system (Euler angles).

The necessity to also control torsional eye position becomes clear if we consider that the same horizontal and vertical rotation of the eye but in different sequence results in a different torsional eye position in space (rotations are not commutative). For example, rotation of first 45 degrees up, then 45 degrees right results in a counterclockwise rotated retinal image; rotation first 45 degrees to the right, then 45 degrees up results in a clockwise rotation of the retinal image. A corrective torsional movement of the eye is therefore required to provide the same orientation of the image on the retina independent from the sequence. As identified more than a hundred years ago by Donders and Listing3,4 all eye positions during a fixation may be described as a rotation around a single axis from a primary eye position (approximately straight ahead). All rotation axes are lying in a single plane perpendicular to the primary position of the eye (Listing’s law) (Figure 24-2).

Dynamics of Eye Movements

Eye movements are needed for five main purposes and hence are performed at different velocities and angular ranges (Table 24-1)*:

a.Saccades: The eye normally performs fast eye movements (saccades) to jump from one target position to the other (fixation). Peak velocities of saccades are somewhat proportional to the amplitude of the saccade ranging from 10 degrees per second (deg/s) (2 mm/s) to 800 deg/s (170 mm/s).3 The duration of saccades depends on the amplitude, ranging from 30 milliseconds (ms) for small saccades of 5 degrees to 100 ms or even more for 20 degrees and above

b.Vestibular eye movement stabilizes the retinal image to avoid blurring due to head movements. Linear (translatory) and rotational acceleration of the head are measured by the vestibular system in the inner ear and lead to slow compensatory eye movements up to approximately. 100 deg/s, (21 mm/s), which are often compensated by faster saccades in the range of 100 deg/s to 400 deg/s, (84 mm/s) in the opposite direction if the amplitude of the movement reaches a certain limit (nystagmus). Also static rotations are introduced. Different orientation of the head toward gravity results in a different torsional eye position (ocular counter-roll) in the order of ±7 degrees14

Figure 24-2. Eye rotations according to Listing’s law.

c.Pursuit and optokinetic movement stabilize the retinal image due to moving objects relative to the eye. A retinal image slip creates again a pursuit eye movement of up to 40 deg/s (8 mm/s) to follow the target and minimize retinal slip.8 Movements with larger amplitudes create also fast saccades in the opposite direction, resulting in an optokinetic nystagmus

d.Vergence eye movement removes retinal disparity introduced by looking at objects at different distances and by accommodation (retinal blur).9 In humans, these are slow eye movements in the order of 10 deg/s (2 mm/s) and are mainly horizontal with amplitudes up to 15 degrees; however, vertical and torsional vergence can be observed by introducing prisms10

e.Miniature movements occur while the subject is trying to maintain fixation on a stationary target. These movements consists of three components: 1) tremor, a high frequency movement with a bandwidth up to 90 hertz (Hz),11 which introduces retinal image movement with velocities in the range of 10 min/s12 in the range 10 to 40 seconds of arc and comparable in size to the smallest cones on the retina (some 24 seconds of arc); 2) comparatively large but slow drift movement with amplitudes in the range of 5 min and velocities in the region of 4 min/s13; and 3) microsaccades in the range of 10 feet and peak velocity in the order of 300 min/s.12 Therefore, even during a perfect fixation, humans perform eye movements at least within a range of approx-

Figure 24-3. Distribution of pupil size changes during laser in-situ keratomileusis (LASIK) surgery for one patient.

imately 0.2 degrees (corresponding to 45 µm on the cornea) and peak velocities of around 5 deg/s (corresponding to 1000 µm/s on the cornea)

Eye Movement Specific to Laser Refractive Surgery

Laser refractive surgery may induce different amounts of eye movements to the amount of eye movement measured during laboratory examination. For example, the patient fixates on a target, which may be difficult to see due to the removal of the flap, or the pharmaceutical dilation of the pupil. The surgeon may also introduce head movements while trying to correct the patient’s drifting eye movements. In addition, the mental state of the patient together with auditory cues of the laser during the procedure create distractions from the fixation task.

Schwiegerling and Snyder14 measured eye movement during LASIK performed with a VISX STAR S2 laser system (Santa Clara, Calif). Videotapes of five surgeries were recorded and the results were analyzed off-line. Eye movement was measured by measuring the displacement of the pupil image in the video-

tapes. Two of these eyes were decentered by approximately 0.25 mm relative to the axis of treatment, and the standard deviation of eye movement overall was 0.10 mm.

Other aspects of the eye, apart from pure rotation of the globe, also affect the accuracy of beam position on the eye. These factors include the movement of certain features within the globe, such as the pupil. Most alignment and tracking techniques rely on the pupil center as the reference point for alignment. The doctor is recommended to manually align the patient on the line of sight, defined as the broken line passing through the entrance pupil and exit pupil, connecting the point of regard to the foveola.15 Most automatic tracking systems take advantage of the high contrast between pupil and iris to track on this feature. However, the pupil is not a fixed point relative to the corneal tissue being ablated.

The pupil center does not dilate evenly and the change of pupil center with pupil diameter cannot be predicted.16,17 A study at SMI measuring 11 patients comparing pupil center relative to fixed markers on the eye between a dilated state (as is common during wavefront measurement) and a natural state (as is common during surgery) showed an average pupil center shift of 0.20 mm and a maximum of 0.60 mm. The change in pupil size between diagnosis and surgery can vary from 8 mm to 3 mm. The natural pupil size during surgery varies significantly less. Figure 24-3 shows the pupil size changes during a typical surgery. Therefore, it can be assumed that the pupil center shift during surgery is also significantly less. Hence, to achieve accurate alignment it is more important to control the axis of alignment between the diagnostic device and the eye-tracking device (ie, pupil center shift between diagnosis and surgery).

REQUIREMENTS OF EYE TRACKER

TO COMPENSATE FOR EYE MOVEMENTS

Given that there is an overall requirement for accuracy of beam placement on the cornea (eg, 0.05 mm) and a known expected amount of eye movement, the design requirements for a system that measures and compensates for eye movements can also be derived. Overall accuracy is a combination of the dynamic accuracy of the system and the static accuracy.

Figure 24-4. Open loop image-based eye tracking system

Dynamic Accuracy

Dynamic accuracy can be defined as the errors induced by the fact that the target is moving. For example, if the eye position is measured completely accurately, but it takes a certain latency period to move the laser beam to the measured eye position, any eye movement during this latency period will contribute to the overall inaccuracy.

Figure 24-4 shows an open loop image-based eye tracking system. Each step illustrated in this diagram contributes to the overall latency between the eye position and the laser beam compensation.

a.The eye is illuminated and the light imaged on a sensor device

b.This image is transferred from the sensor to a processing device

c.The processor calculates the x,y position from the image and sends it to a scanner

d.The scanner sends a signal to rotate a mirror to redirect the laser beam

e.The laser beam fires and hits the cornea

Knowing the type of eye movements to take into account, now the question is what are the necessary requirements—sam- pling rate and delay—for measuring them in the context of refractive surgery. One technique is to use the power spectral density* of eye movements (Figure 24-5)—converted already into movement of the cornea—while a subject fixates for 60 seconds either on a single target or viewing relaxed at a fixed picture similar to the scene observed during surgery, which reflects the extreme situation for eye movements during refractive surgery.

This figure shows that if a fixation cannot be performed accurately, then the main energy of eye movements is found below

Figure 24-5. Power spectral density of 60 second recordings of horizontal eye-movements fixating a point target (black) and observing a fixed scene image similar to the scene during refractive surgery (gray) (sampling-frequency 250 Hz; binwidth 0.49 Hz, 256 bins; using Parzen-windowing). Standard deviation of horizontal eye movement was 66 µm during fixation and 2.7 mm viewing the visual scene.

60 Hz. The above power spectrum also demonstrates that for being able to perform accurate reconstruction of eye movements and avoid aliasing sampling rates of 200 Hz and above (at least 4 to 5 times higher than the significant power spectrum bandwidth) should be used.

These results were confirmed by a second study, in which 250 Hz eye tracking data was used to simulate the amount of error that that would occur for a given overall delay between eye position measurement and laser shot. Figure 24-6 shows the different scenarios for the same eye movement pattern given a varying amount of latency or delay. For an overall latency of 48 ms, 80% of the shots were within 0.065 mm of the target. This error was reduced to 80% better than 0.007 mm, when latency was reduced to 4 ms. To put this data into context, state-of-the-art video–based tracking systems may have an overall delay of less than 8 ms. A system based on a standard NTSC (60 Hz) camera may have a latency of more than 50 ms.

Static Accuracy

The accuracy of the eye position measurement, independent of eye movement, must also be given due consideration. Static inaccuracy can come from either errors in the feature detection (eg, errors in the image or signal processing required to detect the pupil center or edge) or due to the assumptions used in measuring eye movement. As errors in feature detection depend heavily on the individual system, this section will discuss only errors

Figure 24-7. Rotation vs translation of the eye.

For an overall latency of 48 ms, 80% of the shots were within 0.065mm of the target. This error was reduced to 80% better than 0.007 mm, when latency was reduced to 4 ms.

There is no eye tracking system that currently actively differentiates between translation and rotation of the eye. Decentration can be minimized by reducing the active or hot zone of the tracking system and switching the laser off when the eye extends beyond this zone, as is done with passive tracking.

due to the assumptions common to most eye tracking systems— assumptions made in tracking a particular feature, such as the pupil.

The eye can move in six different dimensions—translation in x, y, and z; and rotation around x, y, and z, known as pitch, yaw, and roll, respectively—but it is a common assumption to only consider x and y translation movement. Rotation around the x- and y-axis can be approximated as translation, when projected onto a two-dimensional (2-D) image plane. It is a common trait of all refractive surgery eye trackers to track the pupil or to be exact, the projection of the pupil image onto an image plane. Figure 24-7 illustrates the errors caused by this assumption.

In Figure 24-7A, the eye is perfectly aligned and the center of the pupil is aligned to the corneal center (green cross = target for the laser beam). When the whole eye translates (eg, during a translation of the chair), the center of the pupil will remain aligned with the corneal center. Figure 24-7B shows the same eye, rotated around the y-axis. When viewed by the surgeon through the microscope, this rotation will appear like a translation of the pupil center and the corneal center in x. This is the assumption made by all present refractive eye-tracking systems—that eye movement can be approximated by this projected translation. It can be seen in the zoomed view in the middle, that the pupil center underestimates the movement of the corneal surface, as it is a different distance from the center of rotation of the eye. For a rotation of 0.20 degrees or translation of 0.05 mm, this error is only 0.011 mm (calculated assuming a distance of x between pupil and corneal surface). However, for a rotation of 0.80 degrees or 0.20 mm, this error is 0.046 mm and hence significant.

At present, no eye tracking system actively differentiates between rotation and translation of the eye. However, the error

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Figure 24-8. Torsion between diagnosis and surgery (n = 46 subjects).

due to this assumption can be minimized by minimizing the active or hot zone of the tracking system. When the eye moves beyond a given threshold, the laser is switched off and the eye realigned (passive tracking).

Errors in measuring eye position may also be caused by ignoring translations in z, or changes in height of the eye. This depends on the focal depth of the imaging system and is worsened when the tracking system is not coaxial.

Rotation around z, or torsion of the eye during fixation, is relatively small within ±1 degree. However, torsion during surgery is also caused by events such as the flap cut, wiping or marking the eye, or small changes in head position. As there is a large change in head position between surgery and diagnosis, the majority of torsion error is caused as a constant offset between the sitting up position during diagnosis and the prone position under the laser. Figure 24-8 shows an accumulative graph of the amount of torsion measured using the iris registration between an auto-refractor and a refractive laser.

Editor’s note:

“Locking on” in the proper position is the most important step in laser surgery registration. You can have an excellent tracker, but if it isn’t “locked on” properly, it will be ineffective. To get excellent static accuracy in any system, it is critical that the surgeon “lock on” with the eye centered and fixated.

S. MacRae, MD

PRESENT EYE TRACKING TECHNOLOGY

Eye tracking has been used in a variety of applications but the requirements for laser refractive surgery are quite particular. The eye tracker must be fast, reliable, nonintrusive, work on all iris colors, and be insensitive to alignment lights and to the removal of tissue from the cornea. The main techniques applied in refractive surgery can be classified as either open loop or closed loop tracking systems and as either image-based eye tracking systems or photoelectric-based systems.

Open Loop vs Closed Loop Tracking System

A closed loop system uses the output from the previous measurement as input for the new measurement; hence, it closes the loop. An open loop system does not use information from the previous measurement in the new measurement. These two different control techniques have different affects on both the dynamic requirement of the eye tracking system and the robustness of the eye tracking system.

Strictly speaking, an open loop system does not track or follow the eye. Instead, an open loop system aims to take a snapshot of the eye position as close as possible in time to the laser shot. It is not necessary for an open loop system to follow every eye movement, but instead to have a very small delay between eye position measurement and laser shot. Saccades that occur between laser shots may be safely ignored. Therefore, for an open loop eye tracking system, the most important “speed” factor is delay or latency. A closed loop system must capture every movement of the eye to keep locked onto the eye; therefore, for a closed loop system, the most important factor is frequency. The required frequency to capture the eye movements can be derived from the power spectrum shown in Figure 24-5.

Therefore, for an open loop eye tracking system the most important “speed” factor is delay or latency. A closed loop system must capture every movement of the eye to keep locked onto the eye; therefore, for a closed loop system, the most important factor is frequency. An objective performance comparison should be based on the resulting dynamic accuracy.

As these requirements for the two systems are different, the best way to compare the “speed” of the systems is to compare the resulting dynamic accuracy. The dynamic accuracy of a synchronized 250 hertz (Hz) open-loop system with a delay of 8 ms is shown in Figure 24-6—80% of shots better than 14 µm. The dynamic accuracy of a 4 kilahertz (kHz) closed-loop system is approximately 1 µm, with a peak position error of 30 mm during fast saccades.18

Figure 24-9. Tracked eye with dark pupil tracker for x, y coordinates and ocular torsion using the three-dimensional (3-D) VOG system by SensoMotoric Instruments (Boston, Mass).

Figure 24-11. Detector array for limbus tracking used in IRVision AccuScan 2000 (San Jose, Calif).

The decision whether to use open loop or closed loop tracking also affects the stability or robustness of the eye tracking system. As an open loop system does not use information from the previous measurement, it is unlikely to “lose track” when a doctor’s hand or instrument is placed between the eye and the tracking system.

Image-Based (Digital) Systems

In image-based eye tracking, infrared (IR) light illuminates the eye and IR sensitive cameras acquire the eye image. If the axis of the illumination and the camera differ significantly, the pupil is a sink for IR light and appears dark on the image. (If the IR illumination and camera are close to or on the same optical axis, the IR light is reflected by the retina and the pupil appears light on the image.) The dark pupil approach is more commonly used in refractive surgery, since it is more robust in illumination set-up. Figure 24-9 shows a typical dark pupil-tracking situation with cross hair overlay for pupil center and tilt of cross hair for torsion of the eye around the visual axis.

With more powerful imaging sensors and processing units and more sophisticated algorithms for determining the pupil center and the limbus, video eye tracking is getting increasingly robust and insensitive to a broad range of disturbances, changes of the illumination, and image quality during refractive surgery.

Figure 24-10. Detection of partly occluded eye with image processing using the 3-D VOG system by SensoMotoric Instruments.

The following image (Figure 24-10) shows an example of a partly occluded eye, in which the pupil is still detected.

Image-based tracking also has the advantage of improved static accuracy using enhanced image processing. Measuring both pupil and limbus in the same image can compensate errors caused by pupil center shift. Misalignment caused by ocular cyclotorsion between diagnosis and surgery can also be compensated using iris registration.

Although it is technically feasible to use image-based systems as a closed loop system, most are open loop tracking systems.

Photoelectric-Based (Analog) Systems

Photoelectric analog techniques detect eye movement from changes in reflected light. Either focused spots, slits, or rings of light are projected onto the cornea, and the response from multiple light detectors is analyzed using analog signal processing techniques. Figure 24-11 shows the configuration of one such tracker detector array. Either the pupil/iris border or the limbus is monitored. If the pupil is tracked, it is generally back-lit to maximize contrast and dilated to minimize interference from the procedure itself. The limbus does not suffer these limitations, as it is located further away from the surgical zone.

Because analog methods are used, the frequency response can be very high (eg, 4 kHZ), which lends these techniques to closed loop tracking systems. On the other side, these techniques tend to measure only a limited number of points on the eye, such as the four spots to define the pupil shown in Figure 24-11, compared to the approximately 75,000 points measured by a highspeed video system. This may result in a lower measurement resolution, and limits these systems to 2 diopter (D) translation tracking.

CONCLUSION

Eye trackers are a basic requirement of laser refractive surgery and essential for optimal correction. In the quest—not only for ideal corrections but also for reliable repeatable results for all patients—more accurate centration is required. To be certain that the alignment of the patient’s eye is sufficiently accurate, several factors need to be considered:

•Accurate registration between diagnosis and surgery, including compensation for pupil center shift and cyclotorsion (mean values 0.20 mm and 3.40 degrees measured from clinical trials)

•Pupil size changes and hence pupil center shift during surgery

•Eye movement measured as the translation of the projected image of the pupil (mean 0.25 mm)

•Rotation versus translation (0.00 to 0.25 mm for eye movement between 0 to 1 mm)

•Rotation around z (torsion) during surgery (0 to 1 degree based on a fixating eye in laboratory conditions)

•Height (z translation)

•Dynamic accuracy of the system (80% < 0.014 mm based on high-speed video-based system)

When the typical values for each of these factors (shown in brackets above) are summed together, the goal of 0.45 mm to ensure at least a consistent optical quality after refractive surgery does not seem so modest! Each of these factors must be carefully controlled to ensure that the desired refractive correction is aligned correctly to the eye, accurately enough to ensure a successful and repeatable visual outcome. Present eye tracking systems control some dimensions of alignment and tracking automatically, repeatably, and objectively and have been demonstrated to improve optical outcome.21 Until eye tracking systems can control all of these factors, the clinician should be aware of how each of these considerations can affect the alignment and hence the visual outcome, and actively work to monitor and control the alignment of the patient’s eye. The fully automatic six dimensional high-speed eye alignment system integrated into both the diagnostic and surgical device, however, is the ultimate goal for controlled consistent customized surgery.

Most patients move their eye only minimally during surgery. Therefore, it is important that the laser “locks on” and gets excellent correspondence between the axis and the orientation of the eye measured by wavefront sensor and the axis and orientation of the eye identified by the eye tracker during ablation.

As the science and technology behind refractive surgery and customized ablation improves so too will the eye tracking and alignment systems. The goal of not only an “ideal correction,” but reliable repeatable results for all patients will be reached not just by faster eye trackers, but also by smart imaging technology eye trackers.

ACKNOWLEDGEMENTS

made this work possible, as well as all our colleagues at SensoMotoric Instruments.

REFERENCES

1.Guirao A, Williams DR, Cox IG. Effect of rotation and translation on the expected benefit of an ideal method to correct the eye’s higherorder aberrations. Opt Soc Am A. 2001;18(5).

2.Bueeler M, Mrochen M, Seiler T. Required accuracy of lateral and torsional alignment in aberration sensing and wavefront guided treatments. Proceedings of SPIE. Vol. 4611: 185-196, In: Manns F, Soederberg PG, Ho A, eds. Ophthalmic Technologies XII. 2002:185-196.

3.Donders FC. Beitrag zur Lehre von Bewegungen des menschlichen Auges, Holländische Beiträge zu den anatomischen und psychologischen. Wissenschafte. 1847;1:101-145, 384-386.

4.Listing, JB. Beitrag zu Physiologischen Optik. Vandenhoeck & Ruprecht: Göttingen; 1845.

5.Atchison DA, Smith G. Optics of the Human Eye. Oxford, United Kingdom: Butterworth-Heinemann; 2000.

6.Bahill AT, Clark, MR, Stark L. Glissades-eye movements generated by mismatched components of the saccadic motoneuronal control signal. Mathematical Biosciences. 1975;26:303-318.

7.Miller EF. Counterrolling of the human eyes produced by head tilt with respect to gravity. Acta Otolaryngol. 1962;54:479-501.

8.Honrubia V, Scott BJ, Ward PH. Experimental studies on optokinetic nystagmus. I. Normal cats. Acta Otolaryngol. 1967;65:441-448.

9.Müller J. Zur vergleichenden Physiologie des Gesichtssinnes. Leipzig, Germany: C. Cnobloch; 1826.

10.Helmholtz H. Handbuch der Physiologischen Optik. 1st ed. Voss, Hamburg; 1867. [3rd edition translated by Southall JPC for the Optical Society of America (1924)].

11.Findlay JM. Frequency analysis of human involuntary eye movement. Kybernetik. 1971;8:207-214.

12.Ditchburn RW. The functions of small saccades. Vision Res. 1980;20:271-272.

13.Ditchburn, RW. Eye-movements and Visual Perception. Oxford, United Kingdom: Clarendon Press; 1973.

14.Schwiegerling J, Snyder RW. Eye movement during laser in situ keratomileusis. J Cataract Refract Surg. 2000;26(3):345-351.

15.Applegate RA, Thibos LN, Bradley A, et al. Reference axis selection: subcommittee report of the OSA working group to establish standards for measurement and reporting of optical aberrations of the eye. J Refract Surg. 2000;16:S656-S658.

16.Loewenfeld IE, Newsome DA. Iris mechanics I. Influence of pupil size on dynamics of pupillary movements. Am J Ophthalmol. 1971; 71:347-362.

17.Wilson MA, Campbell MCW, Simonet P. Change of pupil centration with change of illumination and pupil size. Technical Digest on Ophthalmic and Visual Optics. 1991;220-223.

18.Krueger RR. In perspective: eye tracking and Autonomous LADAR radar. J Refract Surg. 1999;15:145-149.

19.Mrochen M, Eldine MS, Kaemmerer M, Seiler T, Hutz W. Improvement in photorefractive corneal laser surgery results using an active eye-tracking system. J Cataract Refract Surg. 2001;27(7): 1000-1006.

INTRODUCTION:

BASIC ASPECTS OF CORNEAL WOUND HEALING

Customized Corneal Ablation

Innovations in an evolving field of refractive surgery have made possible the customization of laser vision correction based on the ability to detect and correct higher-order optical abnormalities (wavefront errors) beyond simple sphere and cylinder. The goal of refractive surgery can now include enhancement of visual performance beyond Snellen acuity of 20/20 by improving retinal image resolution and contrast. This potential for better than the natural best corrected visual acuity has been termed super vision.1 Customized ablation may also improve our ability to treat the patients who are disappointed with the outcomes of their refractive surgeries despite excellent visual acuity because of complaints related to decrease in quality of vision such as glare, halo, and reduced contrast sensitivity. Furthermore, customized corneal ablation will benefit patients whose eyes have irregular or atypical aberrations as the results of postkeratoplasty astigmatism, corneal scarring, decentered ablations, central islands, or lenticular abnormalities.

Wound Healing Responses After Photorefractive

Keratectomy, Laser In-Situ Keratomileusis, and

Laser Epithelial Keratomileusis

Currently, there are three potential refractive surgical procedures that may be used for customized corneal ablation: photorefractive keratectomy (PRK), laser assisted in-situ keratomileusis (LASIK), and laser assisted subepithelial keratomileusis (LASEK) (Figure 25-1). It is not known which procedure will deliver the best fidelity of transmitting the intended correction to the anterior corneal surface (tear film, epithelium, and anterior stroma). This is, in part, due to the qualitative and quantitative differences in three major aspects of wound healing following these procedures: scarring (fibroblast proliferation), epithelial hyperplasia, and collagen deposition.

In PRK, the corneal epithelium is mechanically or photoablatively removed and the anterior cornea is ablated to correct the refractive error.2 This results in the greatest degree of scarring and epithelial hyperplasia. Furthermore, interindividual variability of wound healing is greatest after PRK. In LASIK, a corneal flap is first raised, the underlying stroma is ablated, and

the flap is repositioned.3,4 Despite the relatively small degree of scarring and epithelial hyperplasia after LASIK, the thickness and irregularity of the flap may mitigate the benefits of customized ablation.5 In LASEK, the corneal epithelial adhesion to the underlying Bowman’s layer is chemically reduced by the application of diluted ethanol, the underlying stroma is exposed and ablated, and the epithelial sheet is returned to cover the ablated corneal surface.6-8 It is not known, at the time of this writing, whether the degree of scarring and epithelial hyperplasia are reduced after LASEK as compared to PRK.

Interindividual variability of wound healing is greatest after PRK.

Although the wound healing response after refractive surgery may sometimes be undesirable, it aims to restore the structural integrity of the injured tissues. An important determinant of the visual outcome of patients undergoing refractive surgery is our ability to predict and compensate for the eye’s biologic response to laser surgery. While better understanding of wound healing response is crucial for first generation refractive surgeries, it is indispensable for customized corneal ablation. Biological variability of wound healing confounds and modifies intended results of corneal topography-coupled or wavefront-guided excimer laser ablation.

Clinical Implication of Corneal Wound Healing

Scarring (fibroblast proliferation), cellular hyperplasia, and collagen deposition coexist during the eye’s response to laser; however, they confer distinct effects on refraction and aberration profiles. The scarring or haze leads to a decrease of contrast sensitivity. The irregular deposition of cells or collagen is responsible for treatment regression or generation of higher-order optical aberrations.

The irregular deposition of cells or collagen is responsible for treatment regression or generation of higher-order optical aberrations.

A pharmacologic approach to modifying the complex corneal wound healing cascade has been tried to suppress subepithelial haze and regression after ablation. While available wound heal-

Figure 25-1. Potential effect of corneal wound healing on customized corneal ablation. (A) Customized vs noncustomized ablation: the simulation of PRK includes hypothetical additional ablative pattern (arrows) designed to correct higher-order aberrations. The corresponding noncustomized ablation is indicated by the dotted line. (B) Post-PRK healing: there is epithelial hyperplasia at the site of the custom alterations that tends to mask the custom effect. Stromal remodeling (green broken lines) may also influence these alterations. (C) Post-LASIK healing: In LASIK, the flap may diminish the benefit of customized ablation and contribute to higher-order aberrations. (D) Post-LASEK healing: it is not known whether the degrees of scarring and epithelial hyperplasia are reduced after LASEK as compared to PRK. (Adapted from Wilson SE, Mohan RR, Hong JW, Lee JS, Choi R. The wound healing response after laser in situ keratomileusis and photorefractive keratectomy: elusive control of biological variability and effect on custom laser vision correction. Arch Ophthalmol. 2001;119(6): 889-896.)

ing modulating agents such as corticosteroid and antimetabolites (Mitomycin C) are effective,9-13 they are nonspecific and have many potentially dangerous side effects. Surgical techniques can also be modified to modulate wound healing to clinical advantage. A detailed understanding of various cellular and molecular processes that regulate the wound healing responses to laser will lead to adjunct therapeutic strategies that may mitigate the

Figure 25-2. Light micrographs of the interface between corneal epithelium and stroma (monkey) in areas of excimer laser ablation 6 months after exposure. Top: central area of ablation. Bottom: peripheral area of ablation. Top: Notice the regularity of the basal organization in the central areas as well as a near normal number of cells in the overlying epithelium. Bottom: An increase in the number of layers of cells within the epithelium is seen at the edge of the ablation. The arrow indicates the direction of the lesion center. Bars = 50 microns (µm). (Courtesy of Marshall J, Trokel SL, Rothery S, Krueger RR. Long-term healing of the central cornea after photorefractive keratectomy using an excimer laser. Ophthalmology. 1988;95(10):1411-1421.)

adverse effects and more precisely modulate refractive outcomes. With these insights, surgeons can confidently plan a laser ablation profile, knowing that the results will be more predictable and reproducible. We can then truly realize the worthy goal of “super vision” in the unaided eyes.

EPITHELIAL WOUND HEALING

Following 6 millimeter (mm) excimer ablation of the human cornea, epithelial cells migrate over the wound within 1 to 3 days.14-16 Data from studies of animal and human corneal wound healing following excimer laser keratectomy have shown that there is a tendency for the epithelium to undergo hyperplasia over the ablation bed, which is more pronounced in nontapered ablations (Figure 25-2).14,17-24 Additionally, the epithelial surface does not totally reflect surface irregularities of the stromal ablation bed.20-23

There is a tendency for the epithelium to undergo hyperplasia over the ablation bed, which is more pronounced in nontapered ablations.

In epithelial defects that do not involve the basement membrane, reformation of corneal epithelial adhesion promptly follows epithelial resurfacing.24,25 Adhesion structure reformation, however, is delayed for up to 2 to 3 months following keratecto-

my wounds that go below the basement membrane.25,26 Electron microscopy (EM) and immunolocalization of hemidesmosomes, basement membrane, and anchoring fibril components provide evidence of synchronous reappearance of the adhesion structures following keratectomy wounds.26-35 Postexcimer EM analyses of corneal epithelial basement membrane zones in monkey eyes have shown discontinuities, duplication, and thickening of the basement membrane as late as 18 months postoperatively.2,14,17,18,36,37 Normalization of the basement membrane and hemidesmosomal attachments were seen only at 9 months after wounding.

Data from keratoplasty specimens38 showed that, after excimer laser, 29% of cells had normal basement membrane at 6 months, compared with 86% at 15 months. Multilamellar basement membrane was seen in one cornea. Anchoring fibrils were initially decreased but increased gradually after 6 months. Irregular collagen peaked at 9 months, which coincided with the reduction of keratocytes adjacent to the wound. These data corroborate the findings of previous studies and suggest that significant alterations of corneal epithelial adhesion and basement membrane persist after 6 months and do not normalize until 1 year.

Laminin, present in normal corneas in and below the basement membrane, was immunolocalized to the anterior stroma as early as 1 week after wounding.14 It concentrated in the basement membrane zone by 3 months and continued to show discontinuities as late as 18 months after excimer wounding. Type III collagen, ordinarily seen in corneal scars but not in normal cornea, began to appear below the epithelium 3 weeks postoperatively and persist in the anterior stroma.

Bowman’s layer, a cellular, fibrotic collagen layer, does not regenerate once it’s ablated, and its edges retain their tapered appearance after a graded ablation. The exact physiologic role of Bowman’s layer is not clear. A long-standing, but experimentally unproven, hypothesis has been that this layer provides an important contribution to the biomechanical stability of the cornea. Histologically, this point of view was easily accepted because the collagen fibers in Bowman’s layer are omnidirectionally oriented and strongly interwoven. However, experiments of uniaxial stress-strain analysis performed on the human cadaver eyes showed that Bowman’s layer does not contribute significantly to corneal stability.39

STROMAL WOUND HEALING

Phases of Stromal Wound Healing

Concomitant with the epithelial wound healing, the process of stromal wound healing commences but often lasts for a longer time period. Stromal wound healing occurs in four phases (Figure 25-3). In the first phase, the keratocytes adjacent to the area of epithelial debridement undergo apoptosis, leaving a zone devoid of cells. This cell death has been suggested to initiate the healing response.40,41

In the second phase, the keratocytes immediately adjacent to the area of cell death proliferate to repopulate the wound area. In rat corneas, proliferation occurs 24 to 48 hours after wounding. As part of the second phase of stromal wound healing, the keratocytes transform into fibroblasts and migrate into the wound area. This migration may take up to a week. Transformation of keratocytes to fibroblasts can be visualized at the molecular level

as reorganization of the actin cytoskeleton (with development of stress fibers and focal adhesion structures) and activation of new genes encoding extracellular matrix (ECM) components such as fibronectin, ECM adhesion molecule, 5 integrin, ECM-degrad- ing metalloproteins (MMPs), and cytokines.

This same transition occurs when keratocytes are isolated from the corneal stroma and cultured in serum-containing medium; by the time these cells are subcultured, they have acquired the fibroblast phenotype. The migratory repair fibroblasts (fila- mentous-actin positive) are elongated, spindle shaped, highly reflective and are present in the wound edge and within the wound. Repair fibroblasts turn on the synthesis of the 5 integrin chain that results in formation of the 5b1 integrin heterodimer, the classic fibronectin receptor. This occurs concomitant with deposition of fibronectin in the wound area. Synthesis of dermatan sulfate proteoglycan increases whereas lumican synthesis decreases. In addition to depositing extracellular matrix, repair fibroblasts synthesize several matrix MMPs including MMP-1, - 2, -3, and -9, and -14 (MT1-MMP).

In addition to depositing extracellular matrix, repair fibroblasts synthesize several MMPs.

Freshly isolated keratocytes differ from subcultured cells, or from migratory repair fibroblasts, in their incompetence to synthesize collagenase in response to treatment with agents that stimulate remodeling of the actin cytoskeleton, such as phorbol myristate acetate (PMA) or cytochalasin B (CB).42 Collagenase expression requires activation of an autocrine interleukin (IL) 1 feedback loop, which in turn requires two different stimuli. Agents such as CB or PMA provide an initial stimulus. However, in order for the positive feedback amplification to occur, cells must subsequently be responsive to the stimulation from their self-generated IL-1 (autocrine pathway). The incompetence for expression of IL-1 by fresh corneal stromal cells is the result of failure to activate the transcription factor NF-kB. Interestingly, NF-kB regulates many cell functions with potential to disturb corneal structure, including expression of inflammatory, stress, degradative proteinase genes, apoptosis, and cell replication. Therefore, NF-kB regulatory pathway likely represents an important mechanism for maintaining corneal homeostasis and preserving functions.43

In the third phase of stromal wound healing, fibroblasts may be transformed into myofibroblasts (evidenced by smooth muscle actin staining). This occurs primarily after incisional wounds. Myofibroblasts appear as stellate cells, are highly reflective, but are limited to within the wound area. The extent of transformation into myofibroblasts seems to be dependent on the type of wound. In general, gaping wounds and wounds that remove Bowman’s membrane result in greater myofibroblast generation than wounds that do not penetrate Bowman’s layer. Myofibroblast transformation may take up to a month to become apparent.

In general, gaping wounds and wounds that remove Bowman’s membrane result in greater myofibroblast generation than wounds that do not penetrate Bowman’s layer.