1.

Introduction

Although laser-assisted in situ keratomileusis (LASIK) has become increasingly popular for myopia correction, excimer laser–based photorefractive keratectomy (PRK) remains a valuable technique for the highly myopic patient or individual with relatively thin corneas. Nevertheless, the post-PRK wound healing process may lead to the development of corneal haze, resulting in impaired vision.¹ The opacity resulting from the post-operative wound healing process usually develops $1\text{month}$ after the PRK procedure and reaches peak severity $3\text{months}$ post-surgery. Both epithelial-scraping procedures and PRK have been reported to induce cell apoptosis of keratocytes,^{2, 3} with transepithelial PRK inducing less anterior keratocyte apoptosis than laser-scraped PRK.⁴ These results suggest that only the wounds traversing Bowman’s layer into the stroma will induce keratocyte activation, where the keratocytes are transformed into spindle-shaped myofibroblasts.^{2, 3, 5} The transformation of keratocytes into myofibroblasts is characterized by a larger cell appearance, progressive deposition of new extracellular matrix (ECM), and the expression of $\alpha$ smooth muscle actin $\left(\alpha \text{-}\mathrm{SMA}\right)$ .^{2, 3, 5, 6, 7, 8, 9, 10, 11, 12} The extent of keratocyte apoptosis and activation also depends on the depth of ablation in the surgery.^{11, 13} A possible explanation is that more intensive corneal haze is caused by more severe neural damage from a deeper penetrating wound.¹⁴ It has been proposed that inflammation, epithelial hyperplasia, keratocyte activation, and deposition of new ECM are the factors responsible for increased opacity in post-PRK corneal stroma. ^{15, 16, 17}

Recently, mitomycin C (MMC) has been reported as a myofibroblast suppressor to modulate corneal wound healing and prevent corneal haze and regression.^{18, 19, 20} MMC, an alkylating antibiotic derived from Streptomyces caespitosus, suppresses the proliferation of rapidly growing cells by inhibiting DNA synthesis.¹⁹ The reports discussing MMC-modulated wound healing usually emphasize keratocyte apoptosis, repopulation, and myofibroblastic transformation. However, the tissue responses after PRK remain to be clarified. For example, the decrease in corneal clarity can result from the different alignment of the regenerated collagen during the wound healing process. These changes in corneal collagen cannot be effectively visualized by standard optical imaging techniques, due to the high transparency of cornea.

In recent years, advances in multiphoton microscopy have rendered label-free imaging of the ocular surface, including the cornea and sclera, a reality.^{21, 22, 23} Advantages of this technique include excellent axial-depth penetration and discrimination with significantly reduced photodamage. In this approach, cellular architecture can be visualized by multiphoton autofluorescence (MAF) imaging, while corneal collagen can be imaged using the intrinsic second-harmonic generation (SHG) signal. In this study, we used multiphoton microscopy in conjunction with traditional histology and immunohistology to investigate the wound healing process in rabbit corneas following PRK procedure. To address the potentiality of noninvasively observing myofibroblast formation, we correlate the number of activated keratocytes identified by bright MAF with the number of $\alpha$ -SMA positive myofibroblasts in immunohistochemistry (IHC) imaging. For possible future clinical applications, the SHG signal used to monitor stromal collagen alteration is detected in the backward direction. The effectiveness of post-PRK MMC treatment in suppressing myofibroblast transformation and collagen deposition is also evaluated.

2.

Materials and Methods

2.4.

Quantitative Analysis

To determine the density of myofibroblasts in the anterior stroma, the number of $\alpha$ -SMA positive cells (myofibroblasts) in the immunostained histological sections and the number of activated keratocytes identified by bright MAF in the multiphoton images were separately counted by two investigators of this study. For our purpose, the anterior stroma is defined as the upper $1∕3$ of the cornea where the keratocytes are concentrated.¹⁰ For calculating the myofibroblast density in the immunohistological section, five nonoverlapped, $100\times 100\mu {\mathrm{m}}^2$ regions selected from the anterior region in each cornea were used for cell number counting. Since the immunostained images display a cross-section view of the whole cornea thickness, averaging over the five regions accounts for the density variation due to depth. To correlate activated keratocyte in multiphoton imaging to the myofibroblast number in an immunohistochemical slice, we measured the number of MAF positive keratocytes in the en face multiphoton images $(400\times 400\mu {\mathrm{m}}^2)$ in the anterior stroma.

Using the SHG images, we can quantify the collagen produced post-PRK procedure. The newly generated collagen can be morphologically identified by its fiber feature in the SHG images. Figure 1 shows the typical fiber morphology at varying depth for a normal rabbit cornea, while in Fig. 2 (row II), we see a $12\text{-week}$ post-PRK rabbit cornea displaying newly generated collagen that is morphologically different from that of normal collagen. By calculating the ratio of the thickness of newly regenerated collagen by the full stroma thickness, we obtained the percentage of regenerated collagen in stroma. In the MTS histological sections, new collagen depositions with increased fibrosis appear in dark blue. Measurement of the thickness of the dark blue region can be correlated with the results from the SHG images.

Fig. 1

SHG images of normal rabbit cornea. (a) SHG imaging across entire rabbit cornea. (b) 3-D imaging stack obtained in the central cornea. (c) to (f) En face sections in central cornea at the depths of 0, 180, 410, and $820\mu \mathrm{m}$ .

Fig. 2

MAF and SHG imaging of corneas with and without MMC treatment after PRK at day 0 and week 12 [(a) day 0, without MMC; (b) day 0, with MMC; (c) week 12 without MMC; (d) week 12 with MMC] Row I: 3-D reconstructions of the corneas. Rows II to V: En face SHG images at stromal surface, depth of $180\mu \mathrm{m}$ , middle stroma, and stromal bottom. The red pseudo color and gray scale represent MAF and SHG, respectively. (Color online only.)

3.

Results and Discussion

A large-area backward SHG image across the entire normal rabbit cornea is shown in Fig. 1. An image stack and depth-resolved images at the central corneal region are also shown in Figs. 1, 1, 1, 1, 1. In Fig. 1, a global concentric pattern of the stromal collagen can be observed, which agrees with earlier results observed.²⁵ The image stack and the depth-resolved images in Figs. 1, 1, 1, 1, 1 show that short and interlacing fibers can be observed on the stromal surface, while global organization of collagen can be imaged in deeper stroma. These observed collagen fiber patterns from rabbit corneas are similar to earlier observation in bovine, porcine, and mice corneas.^{25, 26, 28}

MAF and SHG imaging of corneas with and without MMC treatment after the PRK procedure at day 0 and week 12 are shown in Fig. 2 [(a): day 0, without MMC; (b): day 0, with MMC; (c): week 12, without MMC; (d): week 12, with MMC]. The 3-D reconstructions of corneas are shown in row I of Fig. 2. The MAF and SHG signals are represented by red and gray pseudo-color, respectively. One key feature worthy of notice is that MAF imaging allows the imaging and identification of the epithelium and activated keratocytes. In Figs. 2(a-I) and 2(b-I) MAF positive kerotocytes can be observed throughout the full thickness of corneas, with and without MMC treatment. However, in Figs. 2(c-I) and 2(d-I), the MAF positive keratocytes were found to gather in the anterior stroma as the yellow arrows indicated. The SHG images in row II to row V in Fig. 2 demonstrate stromal structure of post-PRK corneas at increasing depths [II: the top stroma $\left(0\mu \mathrm{m}\right)$ or the subepithelial region of cornea; III: at $180\mu \mathrm{m}$ ; IV: the middle stroma; V: the bottom stroma]. In Figs. 2(a-II) and 2(b-II), normal stromal structure of short and interlacing fibers was not found on the top of stroma. At $12\text{weeks}$ , reticulate, newly generated collagen can be observed in the subepithelial region of corneas without MMC treatment [Fig. 2(c-II)]. The reticulate structure was absent in the week 12 of MMC-treated corneas. However, except for the structural abnormality observed in Fig. 2(d-II), SHG imaging of stroma below $180\mu \mathrm{m}$ reveals similar structural morphology as that found in normal corneas. These results suggest that post-PRK wound healing and MMC treatment do not alter the collagen structure in the stroma bed at depth below $180\mu \mathrm{m}$ .

To focus on the variation at the wounding site, MAF and SHG imaging in the subepithelial region of corneas after different healing times are shown in Fig. 3 . Specifically, the difference between post-PRK cornea with and without MMC are displayed using MAF and SHG imaging, for day 0 and weeks 1, 2, 4, 8, and 12 post-PRK procedure. Selected regions of interests (ROIs) in the SHG images of Fig. 3 (white dashed squares) are magnified and shown in Fig. 4 . Unevenness on the surface is still observed (Fig. 4, open arrows). On the other hand, reticular collagen structure can be observed in corneas without MMC treatment since week 2. Structures of the reticular network, indicated by solid arrows in the magnified images in Fig. 4, suggest that they are the newly deposited collagen. As the healing progressed, the newly generated collagen networks became increasingly dense (Fig. 4; week 12). Moreover, we observe that the stromal bed containing the newly deposited collagen became increasing thick as well. This trend is verified by histological observation (Fig. 5 ). Figure 5 shows the histology with MTS of a post-PRK cornea without MMC treatment, while Fig. 5 shows the similarly stained normal cornea without any surgery. The yellow arrows in Fig. 5 indicate a dark blue region that is the region with enhanced collagen fibrosis, identified by the reticulate structures of collagen fibers in the SHG images in Fig. 3 and Fig. 4.

Fig. 3

MAF and SHG images of stromal surface with and without MMC treatment at day 0 and weeks 1, 2, 4, 8, and 12 following PRK procedure. Selected ROIs enclosed by white dashed lines are magnified in Fig. 4. The solid arrows indicate dendritic and activated keratocytes in response to wounding. The open arrows indicate spindle-shaped, activated keratocytes.

Fig. 4

Magnified SHG images of selected areas in Fig. 3. The solid arrows indicate newly deposited collagen networks. The open arrows point out the unevenness of the stromal surface after PRK treatment.

Fig. 5

Masson’s trichrome stain of (a) week 12 post-PRK cornea without MMC treatment and (b) normal rabbit cornea without surgery. The dark blue regions, indicated by yellow arrows, are the regions with collagen fibrosis. (Color online only.)

To quantify our observation, we measured the ratio of the thickness of newly deposited collagen over the entire stromal bed in both the SHG imaging and MTS histology results (Table 1 ). The results in Table 1 show that the collagen deposition rate calculated from SHG and MTS imaging is comparable, as both methods show that MMC effectively reduced the collagen deposition after the PRK procedure. Our results show that SHG imaging provides a label-free and less invasive method for measuring the presence of newly deposited collagen than the histological method. In addition, the SHG images and the MAF results in Fig. 3 show the interesting feature of increased level of keratocyte autofluorescence after the PRK procedure. As the solid white arrows in the day 0 MAF images indicate, dendritic keratocyte with bright MAF were observed in corneas both with and without MMC treatment. The increase in keratocyte MAF may be attributed to the elevated level of intracellular metabolism in response to external stress. This effect has been previously reported, and in our case, we can attribute keratocyte MAF activation to the wound healing from the PRK procedure.^{31, 32} The open arrows in the MAF images show that keratocytes transformed into spindle-shaped myofibroblasts after week 1.^{33, 34} To quantify and correlate our observation with histological results, temporal profiles of keratocyte activation from MAF imaging and $\alpha$ -SMA–positive myofibroblasts from IHC imaging were determined (Fig. 6 ). Initially, the numbers of MAF-positive, activated keratocytes precipitately increased and peaked at week 2. Then, keratocyte activation subsided after week 4. In addition, the average number of activated keratocytes in the anterior region of corneas without MMC treatment was twice as much as in the MMC-treated corneas at week 2 and week 4. Moreover, the number of activated keratocytes in MMC-treated corneas is always less than that in corneas without the MMC treatment (Fig. 6). In comparison, the temporal profile of IHC-identified myofibroblasts has a very similar trend to the MAF-positive, activated keratocytes (Fig. 7 ). Our temporal profile of the myofibroblast population is very close to earlier observation in confocal microscopy and immunohistology.^{5, 10, 13, 34, 35} These results show that MMC effectively suppressed the appearance of myofibroblasts in IHC images and activated keratocytes in MAF. The observed suppression of the number of myofibroblasts or activated keratocytes by MMC is also consistent with earlier investigations, which showed that MMC reduces the myofibroblast formation rate by increasing cell apoptosis.^{20, 35}

Fig. 6

Temporal analysis of the population of activated keratocytes with detectable MAF.

Fig. 7

Correlation between the densities of activated keratocytes in MAF imaging and myofibroblasts in IHC images. $R^2$ is the linear correlation coefficient.

Table 1

Percentage of newly generated stroma thickness relative to residual stromal bed. The errors originate from standard deviation of sample means.

We also found that the number of activated keratocytes in MAF is linearly correlated to the number of myofibroblasts in IHC images (Fig. 7). This result suggests that activated keratocytes with detectable MAF in multiphoton imaging can be used to evaluate the amount of myofibroblast related to scar formation and post-PRK corneal haze. The strength of the MAF signal, however, is relatively unaffected by the number of activated keratocytes. A possible explanation is that the keratocyte activation process occurs much more quickly than the image acquisition time, so only the maximum MAF signal is observed from the activated keratocytes.

4.

Conclusion

In conclusion, we demonstrated that multiphoton microscopy can be used to monitor the alteration in extracellular matrix (ECM) and keratocyte activation in post-PRK corneas. Our results show that MMC decreases the collagen deposition rate during the wound healing process by suppressing the number of myofibroblasts. While similar information relating myofibroblasts and the ECM can be obtained with IHC histology, it is invasive and not suitable for use on a live patient. Clinically friendly reflective confocal microscopy allows the detection of activated keratocytes but cannot be used to monitor the ECM. Since both the SHG and MAF imaging modalities we demonstrated in this work are label-free, our results show that the wound healing process from PRK procedures can be visualized and quantitatively evaluated with minimum invasion.