Cornea and External Disease and Refractive Service, Singapore Eye Research Institute, Singapore, Singapore
Singapore National Eye Centre, Singapore, Singapore
Department of Clinical Sciences, Duke-NUS Graduate Medical School, Singapore, Singapore
20.4 Apoptosis Detection
Refractive lenticule extraction (ReLEx) is a new corneal refractive procedure in which the femtosecond (FS) laser cuts an intrastromal lenticule corresponding to the patients’ refractive correction, without the use of microkeratome or excimer laser . The lenticule is then removed through a surface incision of varying size depending on whether the femtosecond lenticule extraction (FLEx)  or small incision lenticule extraction (SMILE) [3, 4] procedure is being performed.
A significant advantage of the SMILE form of ReLEx surgery is the flapless nature of the procedure whereby the lenticule is extracted through a small pocket incision, obviating most flap-related complications. Another potential advantage of ReLEx is that it could be a reversible refractive procedure: The removal of the fully intact refractive intrastromal lenticule in situ allows the possibility of reimplantation. However, to achieve this, the keratocyte viability and overall collagen structural integrity of the extracted stroma lenticule must be maintained. The concept of preserving this lenticule for either subsequent reimplantation into the same patient or as allograft donor tissue in other patients has formed the basis of the first reversible laser refractive procedure.
Previous studies have long shown that corneal tissue can be stored using cryopreservation [5–7], although the process of freezing and thawing has also been shown to damage corneal endothelium and stroma [7–9]. However, it is now possible to cryopreserve the extracted lenticule and reimplant it back into the donor cornea as a method of autologous stromal volume restoration. Recent studies have shown that the stromal lenticule can be preserved to remain viable after cryopreservation and thawing . The intrastromal keratocytes remained viable, undifferentiated, and expressed markers typical of keratocytes from fresh tissue .
20.2 Cryopreservation Technique
The developed cryopreservation technique for lenticules was as follows [10, 11]: Extracted lenticules were washed in a phosphate-buffered saline (PBS) buffered antibiotic/antimycotic solution and then transferred into a cryovial and resuspended in 500 μl medium containing 10 % fetal bovine serum (FBS). A stock freezing solution containing 10 % FBS and 20 % dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO), a nontoxic cryoprotectant, was added making up a final volume of 1 ml freezing solution containing 10 % FBS and 10 % DMSO. This helped to prevent intralenticular cell damage during freezing in liquid nitrogen . Freezing of the cryovial containing the stromal lenticule was carried out at a controlled cooling rate within a cryo-container (“Mr. Frosty”; Thermo Fisher Scientific, Roskilde, Denmark) in a −80°C freezer overnight and transferred into liquid nitrogen the following day for long-term storage (1 month). This approach has been shown to reduce the damage caused by intracellular ice formation . After 1 month, the vial with the frozen stromal lenticule was rapidly thawed in a water bath at 37°C and rinsed twice in a PBS solution to remove cryoprotectant agents.
20.3 Ultrastructural Analysis
There was a similar pattern of apoptotic and quiescent keratocytes observed in the fresh and cryopreserved lenticules by transmission electron microscopy (TEM)  (Fig.20.1a). Post cryopreservation, the lenticule collagen fibril architecture was found to be comparable to that of freshly extracted lenticules, with a well-preserved and aligned structure, without fragmented fibrils or areas of collagen disruption. This regular collagen structure and organization was also maintained after thawing (Fig. 20.1b). However, because of tissue hydration after cryopreservation, the lenticule collagen fibril density (CFD) was lower post cryopreservation (from 15.75 ± 1.56 to 12.05 ± 0.62, p = 0.02) although there was no significant change in the number of collagen fibrils (p = 0.09) . Regular collagen architecture is one of the key factors in maintaining cornea transparency . Therefore, the maintenance of regular cornea collagen architecture is important following cryopreservation, if the lenticule is to be considered for reimplantation.
(a) Transmission electron micrographs of stromal lenticule showing keratocytes. A, C: Fresh lenticule. B, D: Cryopreserved lenticule. A, B: Apoptotic keratocytes with chromatin condensation and fragmentation, apoptotic bodies, loss of cytoplasm, and cell shrinkage. C, D: Necrotic keratocyte, with incomplete nuclear membrane and vacuoles in the cytoplasm. Magnification, 8900×. (b) Transmission electron micrographs of the stromal lenticule showing collagen fibrils. A, C: Fresh lenticules. B, D: Cryopreserved lenticules. A, B: Transversal section of collagen fibrils. C, D: Longitudinal section of collagen fibrils. Magnification, 50,000× (Courtesy of Mohamed-Noriega et al. )
20.4 Apoptosis Detection
Following cryopreservation, there were significantly more TUNEL-positive cells and a proportional reduction in the number of DAPI-stained cells in the center of the lenticule compared to the periphery . However, altogether, there were more TUNEL-positive cells located in the periphery than in the center of both fresh and cryopreserved lenticules . This implied that the peripheral damage was produced by FS laser, although the keratocytes located in the center were more susceptible to damage during cryopreservation and thawing process.
20.5 In Vitro Cell Viability and Gene Expression Analysis
Viable keratocytes were able to be cultured from both fresh and cryopreserved lenticules, and there was no difference in cellular morphology or proliferation rates between both groups [10, 13, 14] (Fig. 20.2). This suggests that although dead keratocytes were seen using TEM and TUNEL assay, there were enough viable keratocytes within the cryopreserved lenticules that could be isolated and propagated. Gene expression demonstrated keratocyte-specific markers, human aldehyde dehydrogenase 3A1 (ALDH3A1), and keratocan (KERA) to be found on both cells from fresh and cryopreserved keratocytes . Both of these proteins are involved in the maintenance of corneal transparency [13, 14].
Representative images of cultured keratocytes from ReLEx lenticules. (a, b, e, g) Fresh samples. (c, d, f, h) Cryopreserved samples. (a, c): ReLEx lenticules. (b, d) Free-floating stromal keratocytes following enzymatic digestion for at least 4 h in collagenase. (e, f) Attached keratocytes beginning to elongate into spindle-like fibroblastic cells by day 2 in culture. (g, h) Confluent stromal fibroblasts after 7 days in culture (Courtesy of et al. )
Hence, stromal lenticules extracted from ReLEx has been shown to remain viable after cryopreservation . Although there was a decrease in CFD, the overall collagen architecture was preserved and there was good keratocyte viability. Keratocytes have been shown to be an important contributor for maintenance of corneal transparency, and this may be important if the lenticule is to be reimplanted in the future [13–17]. However, as corneal stromal buttons decellularized of keratocytes have been shown to be viable following host keratocyte migration , the maintenance of overall collagen structural integrity post cryopreservation may be the more important finding.
20.6 Lenticule Reimplantation
The concept of potential reversibility can have significant appeal to patients by offering them the reassurance of being able to restore their corneas to the preoperative state and also allowing other future treatment. Potential uses of lenticule reimplantation include the correction of iatrogenic corneal ectasia, where stromal volume is restored in areas of thinning. This may be combined with collagen cross-linking performed to both the lenticule and host cornea for further structural re-enforcement to further arrest the ectatic process . It may also be used as a means of treating presbyopia, by reimplanting the autologous lenticule reshaped to a +1.5 or +2.0 D power, in the nondominant eye of a previously myopic patient who had undergone refractive surgery to near emmetropia, to create a state of monovision . With informed consent and serology clearance, it may also be used as an allogenic biological intrastromal inlay in the same manner as a synthetic corneal refractive inlay that has demonstrated some promise in the treatment of presbyopia, yet obviating the issues of polymer biocompatibility and complications such as corneal melting, alteration in tear film thickness and corneal topography, corneal erosions, and peri-inlay deposits [21–24].
The proof of concept of the idea of autologous cryopreserved lenticule reimplantation has been demonstrated in rabbit and a long-term monkey model [25, 26]. In the rabbit model, it was demonstrated that lenticule reimplantation restored preoperative corneal thickness and caused minimal corneal haze and wound healing responses in the short term . At 28 days post-reimplantation, the implanted corneas were indistinguishable from unoperated control eyes . In the monkeys, the safety, efficacy, and long-term outcome of autologous, cryopreserved lenticule reimplantation following myopic correction were further evaluated, with an emphasis on determining the potential for reversibility with regard to restoration of corneal thickness, curvature, and refractive status .
The rabbits underwent −6.00D ReLEx (FLEx) correction in one eye with the contralateral eye used as unoperated controls . Stromal lenticules were transferred on to rigid gas permeable (RGP) contact lenses (Bausch & Lomb) with careful attention to maintaining anatomical lenticular orientation. The contact lens was placed in a lens case and cryopreservation technique was similar as described above . Reimplantation of the lenticule was carried out 28 days after initial ReLEx (FLEx) procedure.
Slit lamp photographs showed that corneal clarity progressively improved from day 3 to day 28 following lenticule reimplantation and on day 28 was comparable to before ReLEx (FLEx) surgery (Fig. 20.3a). This was matched by a commensurate reduction in interface reflectivity based on confocal microscopy measurements: The anterior and posterior border of the lenticule showed increased light reflectance and was acellular on day 3 after reimplantation. On day 14, the reflective layer at both interfaces was less prominent and keratocytes were visible, particularly at the posterior interface of the lenticule (Fig. 20.3b). The reflectivity level of the anterior and posterior borders were seen to decrease over the duration of the study, with the intensity of the anterior border decreasing from 117.09 ± 20.67 on day 3 to 83.73 ± 14.15 on day 28 and the posterior border decreasing from 105.15 ± 12.87 on day 3 to 90.09 ± 14.10 on day 28. Significant difference (p < 0.05) was noted between day 3 and control and day 14 and control at both interfaces. The final keratometry following reimplantation was −0.6 ± 0.8 D from the preoperative correction. AS-OCT showed the corneas to be edematous compared to control eyes on day 3 after reimplantation but returned to normal at subsequent time points (Fig. 20.4).
(a) The top panel shows slit lamp photographs of the nonoperated cornea (control) and cornea on days 3, 14, and 28 after lenticule reimplantation. The bottom panel shows retro illumination photographs of the control and postoperative corneas. (b) In vivo confocal micrographs of the corneas on days 3, 14, and 28 after lenticule reimplantation. The top panel shows the anterior border of the lenticule within the reimplanted cornea. The middle panel shows the presence of quiescent keratocytes within the lenticule’s lamellae. The bottom panel shows the posterior interface of the lenticule. Repopulation of the anterior and posterior borders of the lenticule occurs by day 28 (Courtesy of Angunawela et al. )
Confocal microscopy also demonstrated resident keratocytes within the center of the lenticule, which remained quiescent and did not change in morphology and activity from day 3 to 28. There was repopulation of the anterior and posterior lenticular borders by day 28, with increased numbers of keratocytes appearing at the anterior and posterior border. There was no proliferating Ki67-positive cells noted and only a few apoptotic TUNEL-positive cells found within the lenticule on immunohistochemical staining. Together with positive staining for cellular actin as indicated by the relatively strong staining of phalloidin, which is a contractile cytoskeletal element found within the cell body, this implied that repopulation of the lenticular borders occurred through cell migration of adjacent keratocyte rather than from keratocyte proliferation. This is probably partly due to the fact that the lenticule itself contains a viable resident population of cells.
On immunohistochemical staining, no myofibroblasts or fibroblasts were detected in the reimplanted cornea, which was indicated by the absence of α-SMA. Both these cell types are implicated in scarring and haze formation in the cornea [27, 28]. Leukocyte integrin β2 (CD 18) was seen expressed by only a few cell and predominantly found at the interfaces of the lenticule; this is an inflammatory marker and mediator of polymorphonuclear leukocyte (PMN) migration within the corneal stroma. Tenascin-C, which is normally found in corneal epithelial cells and only found in corneal stroma after an injury, was detected within the lenticule and mainly along the anterior border. Fibronectin was expressed along the anterior and posterior borders of the lenticule on day 28. The weak healing stimulus, as seen by the minimal expression of fibronectin and tenascin-C, following lenticule reimplantation and the lack of inflammation are advantageous in maintaining corneal clarity and refractive accuracy following refractive stromal reimplantation procedures.
In the monkey model, 8 eyes were used to study long-term effect of ReLEx, 14 eyes for long-term effect of lenticule reimplantation, and 2 eyes as controls for immunohistochemical analysis . The eyes underwent −6.00D ReLEx (FLEx) myopia correction and the storage and cryopreservation of the extracted lenticule were conducted as described before [10, 25]. Lenticule reimplantation was performed 4 months after ReLEx (FLEx) procedure. Corneal clarity was noted to progressively improve from 2.43 ± 0.53 at day 3 after reimplantation to 2.00 ± 0.58 at week 2, 1.07 ± 0.73 at week 4, and 0.21 ± 0.27 at week 8 and to stabilize at 0.14 ± 0.24 at week 16. There was no significant difference in the clarity of reimplanted corneas on weeks 8 and 16 compared to preoperated corneas. The effectiveness of this technique in reversing the refractive procedure was demonstrated by the restoration of corneal thickness, curvature, and refractive error indices to near preoperative values following lenticule reimplantation: AS-OCT showed significant difference in corneal thickness (p < 0.001) between the corneas pre- and post-ReLEx, but no significant difference between the corneas pre-ReLEx (425.05 ± 30.25 μm) and post-lenticule reimplantation (423.76 ± 36.67 μm). Cornea keratometry showed the cornea became flatter (54.1 ± 2.4D) 16 weeks after ReLEx, but on week 16 post-reimplantation of lenticule, corneas were steepened centrally and keratometry values were similar to preoperative corneas (58.0 ± 1.2D vs 58.6 ± 2.1D, p = 0.506). There was no significant difference in the corneal spherical error pre-ReLEx and post-reimplantation; the spherical error before ReLEx was −1.64 ± 0.56D, becoming +4.29 ± 0.86D at week 16 after −6.00D myopic correction (p < 0.001) which indicates that the eyes were −0.07 ± 0.45D from the intended correction. The refraction was restored to −1.64 ± 0.35D at week 16 after lenticule reimplantation (p = 0.891).