2.1.

Femtosecond Laser System

To increase the ablation rate of the corneal stroma, we have modified the laser system that was previously described in detail.^17,18 First, the laser was switched to the standard pulse duration mode ($280\pm 10\mathrm{fs}$ pulses instead of $200\pm 10\mathrm{fs}$ in the short pulse mode), which is more power efficient. This alteration enabled us to increase the average power of the fundamental laser radiation (1030 nm) up to 5 W at a 50 kHz pulse repetition rate, which in turn resulted in an average UV power up to 400 mW. Second, higher-speed galvanometer scanners (hurrySCAN II7, Scanlab AG) were installed to reduce the time intervals when the laser is switched off during acceleration. Third, the UV pulse generator was equipped with a beam shaper to achieve a near second order super-Gaussian beam profile at the corneal surface (Fig. 1). The beam diameter at the $1{/\mathrm{e}}^2$ intensity level was estimated to be $115\pm 5\mu \mathrm{m}$, which is in good agreement with the $113\pm 10\mu \mathrm{m}$ obtained using the knife-edge method.²³ Assuming the super-Gaussian intensity distribution, the peak fluence with these laser settings was $135\pm 5\mathrm{mJ}/{\mathrm{cm}}^2$, which is close to the value we used in our previous experiments on rabbits.¹⁸

Fig. 1

(a) Image of the UV beam at the ablation plane and (b) intensity distributions along the $x$ and $y$ axes. The CCD camera recorded the fluorescence of a sapphire plate positioned at the ablation plane.

We also modified the scanning pattern to maintain low local heating and to achieve a smooth ablation surface at a higher pulse repetition rate. The tissue was removed by ablating circular layers, each of which consisted of five sublayers. The laser beam was scanned in a raster pattern with the distance between adjacent raster lines in a sublayer of $275\mu \mathrm{m}$ and the distance between adjacent spots in the raster line of $55\mu \mathrm{m}$. Each of the following sublayers was displaced by $55\mu \mathrm{m}$ in the direction perpendicular to the raster line. The layers were rotated with respect to each other (Fig. 2). This arrangement ensured efficient cooling due to heat migration into the depth of the cornea and in the direction perpendicular to the raster line while minimizing the number of lost laser pulses. The myopic ablation of stroma was realized as a stack of ablation layers with varying diameter. In order to minimize the heating of the central part of the cornea, the ablation layer with the smallest diameter was followed by one with the largest diameter, and so on, until the process was completed by two layers of approximately equal diameter.

Fig. 2

The nonstop transepithelial femtosecond ablation procedure. Figure shows the progress of the disappearance of the blue fluorescence as the ablation progresses from the epithelium to the stroma (Video 1 QuickTime, 11.7 MB) [URI: http://dx.doi.org/10.1117/1.JBO.20.5.051037.1].

2.2.

Animals and Surgery

Approval for this study was obtained from the Lithuanian State Food and Veterinary Service (number 0213, issued 2011.02.12). Thirty-three pigmented rabbits aged 3 to 6 months and weighing 1.3 to 3.2 kg were included in the study. Anesthesia was achieved by an intramuscular injection of ketamine hydrochloride (Bioketan, Vetoquinol) ($35\mathrm{mg}/\mathrm{kg}$) and xylazine hydrochloride (Xylazine 2%, Alfasan) ($5\mathrm{mg}/\mathrm{kg}$). After the application of a sterile drape, one drop of topical anesthetic proxymetacaine hydrochloride $5\mathrm{mg}/\mathrm{ml}$ solution (Alcaine, Alcon) was applied, and the eye was immobilized using a vacuum suction ring. Before ablation, another drop of topical anesthetic was administered. At the end of the surgery, the corneas were flushed with a balanced salt solution (BSS) (Distra–Sol, OphconD.Roesee.K.) and dried with a sterile sponge. Dexamethasone/chloramphenicol ointment (OftanDexa-Chlora, Santen) was applied, and a sterile bandage was placed for several hours. During the postoperative period, dexamethasone/chloramphenicol drops (OftanDexa-Chlora, Santen) were applied four times per day for 5 days.

The study involved calibration and three treatment modalities: nonstop TransPRK with femtosecond UV pulses, ablation with excimer pulses, and the modified TransPRK with femtosecond UV pulses.

2.2.2.

Nonstop TransPRK with femtosecond UV pulses

Monolateral nonstop TransPRK at $\sim 110\mu \mathrm{m}$ in depth for an optical zone of 6 mm with a 0.6-mm transition zone was performed in 20 rabbits. We estimated that this would include 60 to $80\mu \mathrm{m}$ of the stromal tissue and that the myopic ablation of $70\mu \mathrm{m}$ central depth within the 6 mm optical zone corresponded to a $\sim 5.0\mathrm{D}$ refraction change. The intended ablation profile is shown in Fig. 3.

Fig. 3

The attempted ablation profile of the TransPRK with femtosecond UV pulses. Eighty percent of the epithelium was ablated without the transition zone, forming a cylinder with abrupt edges. The transition zone was initiated in the deep epithelium and led to the 6 mm optical zone in the stroma, where the myopic ablation was performed.

The central corneal thickness (CCT) was measured using an ultrasound pachymeter (Pocket II, Quantel Medical SA) on a dry surface before and after ablation. An infrared thermal camera (ThermaCAM S65, FLIR Systems, Inc.) was used for the monitoring of the surface heating. The temperature increase presented in graphs corresponds to the hottest spot, which was in the center of the ablated area.

2.3.

Evaluation of the Residual Smoothness

Both eyes of one animal underwent transepithelial excimer ablation, as described above. For comparison, the modified TransPRK with femtosecond UV pulses was performed on the previously untreated eyes of three rabbits after the completion of the follow-up period of 6 months. The animals were humanely killed immediately after the procedure, and their eyes were enucleated for histological examination.

2.4.

Follow-up and Corneal Haze Grading

Two eyes after nonstop TransPRK with femtosecond UV pulses were excluded from the follow-up due to postoperative keratitis; however, their intraoperative data (CCT and ablation rates) were included. Ten rabbits were followed-up for four weeks, and eight were followed-up for up to 6 months. Five eyes after the excimer ablation were followed-up for four weeks, and five for two months. The follow-up period after the modified femtosecond UV ablation was four weeks.

Postoperative slit-lamp biomicroscopy examinations, photography and pachymetry were performed without general anesthesia weekly for four weeks and monthly thereafter. The level of haze in the corneas was graded according to the Fantes scale,³³ which scores the highest degree of haze at grade 4. Corneal haze score data were analyzed using the statistical nonparametric Mann–Whitney test ($p=0.05$), as has been previously reported.¹⁸

2.5.

Corneal Collection and Histology

To evaluate the smoothness, five corneas (two after the excimer and three after the modified TransPRK with femtosecond UV pulses) were obtained immediately after treatment. Five eyes after femtosecond UV $110\mu \mathrm{m}$, five after excimer $150\mu \mathrm{m}$ and four after femtosecond UV $150\mu \mathrm{m}$ ablation were obtained at four weeks after treatment, as previous studies have shown that the haze in rabbit corneas peaks at 1 month after both excimer PRK³⁴ and femtosecond UV ablation.¹⁸ Light microscopy of the specimens stained with hematoxylin and eosin was performed as previously described.¹⁸

3.1.

Ablation Depth and CCT Changes

The results of pachymetric CCT measurements and calculated thickness changes are presented in Table 1.

To evaluate the reproducibility of the epithelial debridement, 11 video-records of the nonstop femtosecond UV ablation process were analyzed. We found that the mean variation of the epithelial thickness within the ablation zone of 6 mm in diameter for each individual rabbit was $\sim 12\%$ of the maximal thickness (4 to $6\mu \mathrm{m}$). No pronounced difference in the debridement time between the center and periphery of the ablated area was evident, and areas with maximal epithelial thickness were distributed randomly. Thickness measurements after epithelial debridement were not possible in the nonstop procedure, however, pachymetry immediately after stromal ablation revealed high reproducibility (see Table 1). The attempted total ablation depth of $110\pm 15\mu \mathrm{m}$ was achieved in 90% of the eyes treated with high-speed nonstop femtosecond UV ablation (Fig. 4). In five corneas treated with the modified femtosecond UV ablation, the average achieved ablation depth ($148.4\pm 15.9\mu \mathrm{m}$) also closely matched the intended ablation depth ($150\mu \mathrm{m}$).

Fig. 4

Statistical distribution of the ablation depth of the nonstop TransPRK with femtosecond UV pulses.

When removing the epithelium during the excimer procedure, the stromal surface was exposed after completing PTK ablation with a depth setting of $55\mu \mathrm{m}$. Although it was more difficult to visually observe the disappearance of the blue fluorescence during this process due to the illumination of the operative field, it was easily detectable on the video. The depth of the PTK in the center of the ablation zone was unexpectedly high, $93.4\pm 12\mu \mathrm{m}$ instead of the expected $55\mu \mathrm{m}$. The total ablation depth was thus $151.4\pm 19.7\mu \mathrm{m}$, significantly greater than planned ($110\mu \mathrm{m}$). There are a number of factors that could lead to a rather large mismatch between attempted and measured corneal ablation depths in the case of excimer ablation. The accuracy of the pachymetry measurements could have been affected by the change in the hydration of the cornea, since some force had to be applied to establish the acoustic contact between the probe and the ablated surface of the stroma. The actual thickness of the stroma could have been reduced due to dehydration because of long treatment time and high absorption of water at the excimer wavelength, and the ablation rate of stroma also could have been affected. Yet another possible source of discrepancy is the fact that the diameter of the treatment zone was only approximately three times the beam diameter, which makes it difficult to achieve a perfectly uniform ablation.

The resultant stromal ablation depth measured after the re-epithelialization in both variations of the ablation procedure with femtosecond UV pulses was found to be as expected; in the eyes ablated with the excimer pulses, this value was $120.8\pm 17.4\mu \mathrm{m}$ instead of the intended $70\mu \mathrm{m}$. At this point, pachymetry measurements should not have been affected by differences in hydration of the stroma, and other possible measurement errors should be similar for all ablation cases. Therefore, we assume that more than the planned stromal tissue was ablated during excimer treatment, and most likely during the PTK stage.

We should also note that significant deviation of the measured ablation depth from the attempted one, which strongly depended on the ablation time, was observed when TransPRK was performed in humans.³⁵

3.2.

Ablation Speed

The CCT data after femtosecond ablation procedures were analyzed, and the ablation rate of the corneal tissue was evaluated. Assuming that the ablation speed of the corneal epithelium and stroma was the same, it could be estimated that $\sim 280\mathrm{pL}$ of corneal tissue per 1 mJ have been removed using the high-speed femtosecond UV laser system. As a result, a refractive change of 1-D in the 6 mm optical zone could be created in $\sim 1.6\mathrm{s}$. Alternatively, evaluation of the refraction change was made by measuring the change of the CCT³⁶ after re-epithelialization and resulted in speed values of $\sim 1.45\mathrm{s}/\mathrm{D}$ for the nonstop procedure and $\sim 1.62\mathrm{s}/\mathrm{D}$ for the modified procedure. In the latter case, the speed was calculated by taking into account only the actual ablation time, i.e., with pauses excluded. The durations of the stages of the ablation are presented in Table 2.

Table 2

Durations of the ablation stages.

3.3.

Temperature Rise

The graphs of the temperature increase for different kinds of treatments are presented in Fig. 5. During the nonstop femtosecond TransPRK, the increase in the maximum corneal surface temperature peaked at the end of the procedure and was $<7°\mathrm{C}$ (Fig. 5). The temporal dependence of the surface temperature was directly related to the laser beam scanning algorithm. Ablation of the epithelium was performed within the area of 6.6 mm in diameter and lasted $\sim 15\mathrm{s}$. After that, the ablation zone was reduced to 6 mm, and myopic ablation started (Fig. 2, see video from the second 15). During the myopic ablation, the average power density was higher in the center of the ablation zone in contrast to the epithelial ablation, where the laser power was distributed uniformly over the ablated area. No pauses for cooling were introduced, and stroma was already preheated due to the heat transfer from the epithelium. These factors caused an additional temperature increase during the stromal ablation.

Fig. 5

Corneal surface temperature changes during the ablation process at the center of the treatment zone. $\mathrm{\Delta }t$ indicates the pause between the epithelial and stromal ablations.

The red line in Fig. 5 (color online) represents the dynamics of the corneal surface temperature during the myopic ablation with excimer laser pulses (epithelial ablation is not included). The recorded increase in the corneal surface temperature was less than 4.5°C during the entire excimer ablation procedure. The lowest corneal surface temperature was registered during the modified femtosecond TransPRK. Due to the pauses, the overall increase in temperature did not exceed 3°C, whereas the maximum temperature increase during the epithelial removal was only 1.5°C (Fig. 5, blue line, color online).

3.4.

Postoperative Complications

Two cases of keratitis were observed after the nonstop TransPRK with femtosecond UV pulses. In one animal, an epithelial defect with corneal infiltration and hemorrhage under the nictitating membrane was noted on the seventh day after surgery. In another case, the healing process was uncomplicated for three weeks, but at week 4, a ring infiltrate in the ablation zone developed. While excimer surgery took place in an operating room, procedures with femtosecond UV pulses were performed in a laboratory room where aseptic conditions could not be completely ensured. Another source of infection could have been a nondisposable suction ring, which was used to immobilize the eyes during the femtosecond UV ablation.

3.5.

Subepithelial Haze Measurements

Figure 6 shows the development of haze after different treatments. No statistically significant difference was found in the data on the corneal haze score after nonstop TransPRK with femtosecond UV pulses and excimer laser ablation during the follow-up period of 1 month. The least intense haze was observed after the modified femtosecond UV treatment. This value was even lower than after the shallower ablation with femtosecond UV pulses.

Fig. 6

Mean corneal central subepithelial haze score dynamics after different treatments.

Comparing the degree of haze in the contralateral eyes of the same animals treated by excimer and the modified TransPRK, we found that the latter treatment induced less haze. Statistical analysis showed a significant difference in the corneal haze at 1 week ($p=0.0135$) and at 1 month ($p=0.0415$) after the modified TransPRK with femtosecond UV pulses compared with haze after the other procedures; however, there were only five rabbits in this group.

3.6.

Histological Examination

In samples taken immediately after ablation, relatively smooth ablated surfaces were observed. Light microscopy revealed no noticeable differences in the surface smoothness after excimer ($150\mu \mathrm{m}$) or femtosecond UV ablation ($150\mu \mathrm{m}$).

At 1 month, all corneas after the femtosecond UV ablation of $110\mu \mathrm{m}$ exhibited normal epithelial thickness, with minor variations in smoothing out the stroma (Fig. 7). In three eyes, possible edema of solitary epitheliocytes was noted. In most of the corneas treated by excimer or the modified femtosecond UV ablation, reduced epithelial thickness was observed. Light microscopy of the specimens with corneal haze demonstrated similar subepithelial fibrous layers in the eyes after different treatments. The deeper stroma and inner layers revealed no visible changes.

Fig. 7

One month after (a) the nonstop TransPRK with high-speed femtosecond UV pulses ($110\mu \mathrm{m}$), (b) excimer pulses ($150\mu \mathrm{m}$), and (c) modified ablation with femtosecond UV pulses ($150\mu \mathrm{m}$). The epithelium is thinner in (b) and (c) (red arrowheads, color online). Black arrowheads indicate subepithelial fibrosis (light microscopy).