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Purpose: The aim of this study was to explore the optimal method of small-incision lenticule extraction (SMILE)-derived lenticules, subjected to long-term preservation using glycerol, under a range of temperatures, and using an array of dehydration agents., Methods: In total, 108 myopic lenticules were collected from patients undergoing the SMILE procedure. Fresh lenticules served as a control group for this study, whereas all other lenticules were separated into 8 groups, which were preserved at 4 different temperatures (room temperature [RT], 4, -20, and -80°C) with or without silica gel in anhydrous glycerol. Evaluated parameters included thickness, transmittance, hematoxylin and eosin staining, transmission electron microscopy, and immunohistochemistry analyses., Results: After a 3-month preservation period, lenticular thickness in these different groups was significantly increased, particularly for samples stored at RT. The mean percentage transmittance of lenticules stored at -80°C with or without silica gel was closest to that of fresh lenticules. Hematoxylin and eosin staining revealed sparsely arranged collagen fibers that were more scattered in preserved lenticules relative to fresh lenticules, particularly in RT samples. Transmission electron microscopy revealed that the fibril bundles densities in lenticules stored at RT were significantly less than those stored at other temperatures. Immunohistochemistry analyses revealed reductions in or loss of CD45 and human leukocyte antigens in all preserved lenticules relative to control samples., Conclusions: Of the tested approaches, the preservation of SMILE-derived lenticules over a 3-month period was optimal at -80°C with or without silica gel in anhydrous glycerol., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose: To report the 3-year clinical outcomes of corneal stromal cell therapy consisting of the intrastromal implantation with autologous adipose-derived adult stem cells (ADASCs), and decellularized or ADASC-recellularized human donor corneal laminas in advanced keratoconus., Methods: Fourteen patients were enrolled in 3 experimental groups. Group 1 (G-1) patients underwent implantation of ADASCs alone (3 × 10⁶ cells/1 mL) (n = 5). Group 2 (G-2) patients received a 120-μm decellularized corneal stroma lamina (n = 5). Group 3 (G-3) patients received a 120-μm lamina recellularized with ADASCs (1 × 10⁶ cells/1 mL) (n = 4). ADASCs were obtained by elective liposuction. Implantation was performed into a femtosecond pocket under topical anesthesia., Results: At 3 years, a significant improvement of 1 to 2 logMAR lines in uncorrected distance visual acuity was observed in all groups. A statistically significant decrease in corrected distance visual acuity was obtained in G-2 and G-3 (P < 0.001) when compared with that of G-1. Rigid contact lens distance visual acuity showed a statistically significant worsening in G-2 (P < 0.001) compared with that of G-1. A statistically significant increase in central corneal thickness was observed in G-2 (P = 0.012) and G-3 (P < 0.001); in the Scheimpflug corneal topography, the thinnest point was observed in G-2 (P = 0.007) and G-3 (P = 0.001) when compared with that of G-1., Conclusions: Intrastromal implantation of ADASCs and decellularized or ADASC-recellularized human corneal stroma laminas did not have complications at 3 years. The technique showed a moderate improvement in (uncorrected distance visual acuity) and (corrected distance visual acuity) in advanced keratoconus., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: To investigate the effect of dehydration on human donor corneal stroma for biobanking., Methods: Epithelium and endothelium of research-grade human donor corneas (n = 12) were scraped off, leaving a bare stroma with attached sclera. The tissues were placed in a large Petri dish prefilled with silica gel in the periphery and stored at room temperature for 14 days. At the end of preservation, the tissues were rehydrated by being submerged in phosphate-buffered saline for 15 minutes. Transparency (using a custom-built device) and thickness (using optical coherence tomography) measurements were recorded before dehydration, after dehydration, and after rehydration of the tissues. Periodic acid-Schiff and alpha-smooth muscle actin (α-SMA) staining before dehydration and after rehydration were performed to determine the presence of keratocytes and expression of α-SMA. Tensile stress-strain before dehydration and after rehydration was performed to evaluate the biomechanical properties., Results: No difference in corneal transparency before dehydration (69.57 ± 6.41%) and after rehydration (67.37 ± 2.82%), P = 0.36, was observed. The corneas were more compact after dehydration. A significant change in thickness between before dehydration (625.8 ± 75.58 μm) and after rehydration (563.6 ± 15.77 μm) stage, P = 0.03, was noticed. The thickness was reduced to 147.6 ± 3.71 μm when dehydrated. Periodic acid-Schiff staining showed presence of stromal keratocytes and α-SMA protein expressed in control, dehydrated, and rehydrated corneas. There was no significant difference in the stiffness between control (27.86 ± 11.65 MPa) and rehydrated corneas (31.46 ± 11.41 MPa)., Conclusions: Human donor corneal stroma can be biobanked for up to 2 weeks in a dehydrated condition without losing their molecular or biomechanical properties after rehydration.

Purpose: To construct an acellular porcine corneal stroma (aPCS) as a human corneal stroma alternative and to further explore its biomechanical properties., Methods: A combination of DNA-RNA enzymes and ultrasound technology was used to strip the native porcine corneal cells. The microstructure of aPCS was observed by H&E staining, DAPI staining, and α-Gal tests. The mechanical properties were detected by a tension machine. Cytotoxicity of aPCS was measured by the MTT assay. The subcutaneous embedding experiment in rats was also used to detect immunity and degradation. The aPCS was transplanted into the rabbit cornea by lamellar keratoplasty, general observations were made at 3 days, 1 week, 1 month, and 3 months after implantation, respectively., Results: The microstructure and mechanical properties of aPCS were not damaged during the decellularization process. The aPCS extracts had no significant cytotoxicity on human corneal stroma cells. Moreover, the subcutaneous embedding experiment in rats demonstrated that aPCS could not be degraded and induced no immune reaction in and around the transplanted discs. More important is that the aPCS reconstructed normal corneal stroma and maintained corneal transparency and thickness, with almost no neovascularization and inflammation at 3 months after surgery., Conclusions: The aPCS prepared in this study had good biocompatibility, safety, and low antigenicity, which has great potential for corneal disease treatment.

Purpose: To measure the refractive index (RI) of the human corneal stroma in vivo using an objective Abbé refractometer (VCH-1) and to determine if RI of the stroma is related to age., Methods: VCH-1 was used to measure RI at the central anterior stroma immediately after lifting the flap in neophyte patients preselected for laser in situ keratomileusis. Surgical procedures continued as preplanned after measuring the RI, and in binocular cases, measurements were taken from the right eye only. Corneal flaps were created using a femtosecond laser system., Results: Mean RI (±SD, range) and age (±SD, range) values were 1.373 (±0.006, 1.358-1.385) and 36.55 (±12.26, 18-74). A significant linear correlation was found between age and RI. Least squares regression lines equating RI with age (x, years) were of the form: RI = 1.36911 + 0.000096x (r = +0.195; n = 115; P = 0.037). Mean RI (± SD) in the 2 age groups separated by the median age (≤34 and ≥35) were 1.371 (±0.007; n = 57) and 1.374 (±0.005; n = 58). The difference was statistically significant (P = 0.018)., Conclusions: RI of the anterior stroma in vivo tends to be increased in older patients when the corneal flap is created using a femtosecond laser device.

Purpose: To measure the refractive index (RI) of the human corneal stroma in vivo using an objective Abbé refractometer (VCH-1) and to determine if RI of the stroma is related to age., Methods: The VCH-1 was calibrated against a standard subjective Abbé refractometer using 7 randomly selected turbid semiliquid media. VCH-1 was used to measure RI at the central midstroma immediately after lifting the flap in neophyte patients preselected for laser-assisted in situ keratomileusis. Surgical procedures continued as preplanned after measuring the RI; in binocular cases, measurements were taken from the right eye only. Flaps were created using a mechanical microkeratome., Results: The VCH-1 was capable of measuring the RIs of all 7 turbid semiliquid media. The average RMS difference between RI estimates according to the VCH-1 and standard subjective Abbé refractometer was 0.003. The mean RI (± SD) of the stroma was 1.369 (± 0.008; range, 1.356-1.390), and the mean age (± SD) of the subjects was 33.7 years (± 8.86; range, 18-56 years). A significant linear correlation was found between age and RI. Least squares regression lines equating RI with age (x, years) was of the form: RI = 1.35711 + 0.00034x (r = +0.383, n = 36, P = 0.011)., Conclusions: RI of the stroma in vivo has a tendency to be increased in older patients when the stroma is resected using a mechanical microkeratome.

Purpose: To examine the transparency characteristics at different times of development in the culture of 2 different types of human corneal stroma substitutes generated by tissue engineering using human fibrin or human fibrin and 0.1% agarose, with human keratocytes entrapped within., Methods: The transparency of these artificial corneal stromas was analyzed after 1, 7, 14, 21, and 28 days of development in culture by determining the scattering and absorption coefficients from the spectral reflectance data of each sample using the Kubelka-Munk equations., Results: The scattering coefficient of both types of bioengineered tissues tended to increase with culture time and wavelength until 550 nm, whereby a slight decrease was observed for longer wavelengths. In general, the spectral distribution of the Kubelka-Munk scattering coefficient of the fibrin-agarose corneal constructs was more stable than that of the fibrin constructs. The absorption coefficient of the human fibrin and fibrin-agarose corneal substitutes tended to decrease with increasing wavelength, and their absolute values were higher for fibrin-agarose than for fibrin scaffolds, especially for short wavelengths. In addition, the spectral transmittance behavior of both types of tissue analyzed was similar to the ones of the theoretical and control corneas, with absolute values above 90% for all wavelengths at 28 days of development., Conclusions: The transparency, scattering, and absorption of both fibrin and fibrin-agarose corneal stroma substitutes indicate that these new biomaterials could be adequate for clinical use.

Purpose: To investigate the influence of graft–host interface on the quality of vision after deep anterior lamellar keratoplasty (DALK)., Methods: Retrospective, nonrandomized, comparative case series. Sixty patients (60 eyes) with advanced keratoconus underwent DALK using the big-bubble technique. Twenty-eight patients had complete stromal excision with Descemet membrane (DM) exposure[DALK with DM baring (DM-DALK)], whereas in 32 cases, a layer of stroma was left adherent to the recipient DM [DALK without DM baring (pre-DM-DALK)]. Twenty-two patients with keratoconus (22 eyes) who underwent penetrating keratoplasty (PK) were chosen for comparison. Main outcome measures were uncorrected visual acuity, best spectacle-corrected visual acuity, low-contrast visual acuity(LCVA), and Pelli-Robson contrast sensitivity., Results: Uncorrected visual acuity was equal in the 3 groups. Median best spectacle–corrected visual acuity was 0.1 logarithm of the minimum angle of resolution (logMAR) in the PK group, compared with 0.06 logMAR in the DM-DALK group (P = 0.66) and 0.12 logMAR in the pre-DM-DALK group (P = 0.016). Pre-DM-DALK patients exhibited worse LCVA than PK (P = 0.029) and DM-DALK patients (P = 0.022), whereas PK and DM-DALK patients showed comparable LCVA (P = 0.974). Pelli-Robson contrast sensitivity was equivalent in PK and DM-DALK groups (P = 0.408) and greater in DM-DALK group than in pre-DM-DALK group (P = 0.038)., Conclusions: In selected patients, quality of vision after DALK was comparable to that after PK when stromal excision was extended to the DM and inferior to vision after PK when layers of stroma were left adherent to the DM. Advances in DALK technique are required to allow easier and repeatable baring of the DM.

Purpose: To evaluate the effect of topical dorzolamide on the intrastromal corneal pressure (ICP) in rabbit corneas in vivo., Methods: This is an interventional prospective study. Topical dorzolamide was applied to 7 eyes of 7 male New Zealand rabbits 3 times daily for 3 consecutive days. The ICP changes were recorded with a pressure transducer connected to the midperipheral cornea. The ICP was measured in the same manner in 7 eyes of 7 male New Zealand rabbits that were treated with artificial tears (control group)., Results: The ICP values averaged -6.2 +/- 3.2, -10 +/- 5.8, and -12.5 +/- 8.7 mm Hg at 15, 30, and 45 minutes in the control group, respectively. In the dorzolamide-treated eyes, the ICP readings were 1.8 +/- 3.4, -0.28 +/- 4.3, and -1.8 +/- 5.3 mm Hg at the same time points, respectively. The differences in the ICP between both groups were significantly different at all time points (P = 0.004, P = 0.005, and P = 0.02)., Conclusions: Measuring ICP is a valid and sensitive method to evaluate in vivo the endothelial function. This method seems to be more sensitive than measuring the central corneal thickness or the corneal deswelling rate in detecting changes in the corneal physiology with the use of topical dorzolamide.

Purpose: The purpose of this study was to evaluate morphological changes induced by corneal collagen cross-linking in a human ex vivo cornea, using confocal, electron, and light microscopy., Methods: The central epithelium was partially removed from ex vivo human corneal buttons. Riboflavin 0.1% solution was applied before ultraviolet A light treatment and then for every 2 minutes for 30 minutes while the corneas were exposed to ultraviolet A light at a wavelength of 370 nm and intensity of 3 mW/cm(2). Each cornea was evaluated using confocal, electron, and light microscopy., Results: Confocal microscopy demonstrated normal-appearing corneas on their initial pretreatment examination, with reduced stromal detail. After treatment, a superficial layer of highly reflective spherical structures (4-10 microm) was observed. Many of these hyperreflective structures appeared up to a depth of 300 microm. The remainder of the corneal stroma and endothelium appeared normal. Electron microscopy showed keratocyte apoptotic changes to a depth of 300 microm. No observable pathologic changes were seen on histology., Conclusions: Based on clinical studies, corneal cross-linking is a promising treatment that appears to be safe and to halt ectatic corneal disease progression. Initial European studies used animal models to extrapolate human protocols. In conjunction with clinical studies, we believe that human ex vivo corneal studies provide a means to evaluate the structural and morphological changes associated with this procedure, within human corneas, in a manner that cannot be accomplished in vivo.

Purpose: To show corneal regeneration in 3 cats that underwent lamellar keratectomy (90%) depth during supradescemetic keratoprosthetic implantation., Methods: Three 2-year-old cats that underwent spontaneous keratoprosthesis extrusion between 15 and 150 days after implanting a supradescemetic prosthesis into their right eyes were studied. Corneal structures and stroma thickness were evaluated by slit-lamp photographs, pachymetry, and confocal microscopy. Regenerated corneal epithelial cells, stroma matrix, and keratocyte morphology were studied with histology and transmission electron microscopy. Epithelial and stromal cell immunocharacterization was performed., Results: Corneas progressively regained normal thickness and improved clarity within 40 to 60 days. Slit-lamp photographs and pachymetry showed gains in stromal thickness until 600 microm or more. In vivo confocal microscopy showed the restoration of normal epithelium and stroma in all cats. Corneal nerves were seen in the regenerated stroma of 2 cats. Immunostaining showed absent alpha-smooth muscle actin (SMA) expression and a keratin K3-expressing epithelium. Electron microscopy showed regeneration of normal epithelium with a well-formed basement membrane, organized corneal lamellae, and the presence of normal keratocytes., Conclusion: Felines are capable of regenerating corneal structures including epithelium and reinnervated stroma matrix after deep lamellar keratectomy. The use of feline models in corneal keratoprosthesis is therefore questionable.

The purpose of this study was to obtain further information on the viability of organ-cultured human cornea. We thus used a specific staining method for succinate dehydrogenase (SDH), which is located in the membrane system of vital mitochondria. We examined fresh and long-term-cultured human corneas. After an initial incubation period in dextran-free culture medium, corneas were stored in a medium containing dextran. With respect to different appearances of the SDH staining, minimal essential medium without dextran seems to have a positive effect on the condition of epithelial cells. After renewal of the medium, keratocytes showed a brief improvement followed by a delayed deterioration, while the endothelial cells were severely damaged. However, best results for all three cell types were observed on the fourth day in a medium containing dextran. We therefore conclude that these corneas were best suited for transplantation.

Photorefractive keratectomy with the 193-nm excimer laser is one of the most promising innovations in refractive surgery. However, routine clinical application is hindered by two main obstacles, i.e., postoperative subepithelial opacity and possible regression of the refractive result. Because we have previously demonstrated beneficial effects of basic fibroblast growth factor (bFGF) on corneal epithelial healing in different in vivo models, we investigated the influence on both epithelial and stromal healing of topical bFGF after excimer laser keratomileusis in a rabbit model. After 7-mm circular deepithelialization, the eyes of 24 New Zealand White rabbits received identical deep stromal laser ablations (depth 50 microns, 6 D, diameter 5 mm) and were randomly assigned to one of four treatment groups [control (phosphate-buffered saline, PBS), bFGF 10 micrograms/application, dexamethasone 0.1%, bFGF + dexamethasone, n = 12 eyes/group]. All treatments were administered four times daily--bFGF until complete epithelial healing, dexamethasone and PBS until 3 months postsurgery. Wound surface regression on time was determined by means of computer-assisted image analysis of fluorescein-stained corneas. Corneal opacity was observed biomicroscopically and graded using a previously established scoring system. After bFGF application for 2-3 days only, a highly significant acceleration in epithelial wound healing speed was found compared with the rate of the other three treatment groups (p < 0.001). A combined therapy (bFGF + steroid) had no effect on the healing rate. Compared with control values, mean scores for subepithelial haze in the other groups were found to be nearly 50% lower during the first 2 postoperative months.(ABSTRACT TRUNCATED AT 250 WORDS)

Patients healing from lamellar keratorefractive surgeries invariably experience postoperative corneal haze. In our lamellar keratoplasty (LKP) rabbit model, visual recovery is concomitant with the invasion of the grafted tissue by viable keratocytes from adjacent host tissue, and the return of corneal clarity is attributed to the remodelling of the stromal tissue by these keratocytes. We used synchrotron x-ray diffraction techniques to elucidate the ultrastructure of the stromal collagen fibrils in rabbit corneas at various time points in the 3 months after LKP surgery. Lenticules were frozen in 50% of the cases. The average spacing of the collagen molecules, which constitute the stromal fibrils, remains unchanged by LKP in both frozen (1.69-1.73 nm) and nonfrozen (1.58-1.75 nm) cases. In the nonfrozen case, the collagen fibril diameters are initially slightly larger than normal (38.3-41.9 nm) but have receded by 2 months postoperatively [similar to the frozen cases (37.7-41.9 nm)]. The post-LKP spacing of the collagen fibrils in the nonfrozen corneas is unremarkable (56.2-69.2 nm). In contrast, the increased collagen interfibrillar spacing in the frozen case is considerable (56.7 to > 94.6 nm) and variable up to 21 days postoperatively. Because the changes in interfibrillar spacing did not always mirror pachymetry changes in the frozen cases, we suspect occasional graft malapposition or the formation of intrastromal, fluid-filled, collagen-free "lakes."

The cave-dwelling Typhlotriton spelaeus larvas live in daylight. The larvas undergo a metamorphosis when the thyroxin level (T3 and T4) increases. They leave aquatic life for terrestrial and subterranean aphotic life. The larval eyes are normal and show good vision response to tests. On the contrary, in the oldest larva and the adult, the eyes are small with poor vision or no vision at all. The lens can disappear but in any case the eyelids grow over the eye. The retinal degeneration takes place during and after metamorphosis. The admitted statement that "after metamorphosis . . . the developing lids close over the eye and invade the cornea" is not confirmed concerning the invasion, because it is now evident that the eyelids separate spatially from the residual cornea. Consequently, the fibroblasts of the eyelids are not able to invade the corneal stroma. In fact, the periocular tissues do not invade the cornea but invade the eyelids' tissues. We are able to confirm that the development of the larval eyes is complete and apparently normal. After metamorphosis, the eyes decrease in size. The cornea "sinks" with the eye into orbit tissues. The eyelids cover the eye and are themselves replaced by a supraocular skin. This fits the description of the human cryptophthalmia. We also demonstrate that even in the degenerate eye the residual cornea retains the main structure of a normal salamander cornea. The preservation of the endothelium and of the Descemet membrane is exceptional in blind cave-dwelling vertebrates because in the other degenerate corneas the edema of the stroma depends on the spatial disjunction of the underepithelial stroma and the supraendothelial stroma. Perhaps, the very advanced development of the eye and the late start of the degenerative processes could explain the relative preservation of the cornea. Thus, the arguments put forward tend to prove that the thyroxinic metamorphosis sets off the growth and fusion of the eyelids but that the eye degeneration is related to hereditary processes.

Endocytosis by keratocytes (corneal fibroblasts) is an important part of the host defense system. To investigate the long-term fate of endocytosed materials, we injected polystyrene latex beads into the corneal stroma of four rabbits. The corneal stroma was observed under a transmission electron microscope 4 and 800 days after the injection. After 4 days, the beads were found not only between the collagen fibers of the stroma, but also in some keratocytes. After 800 days, no extracellular beads were seen, but endocytosed beads remained, surrounded by limiting membranes, in the cytoplasm of keratocytes. These observations demonstrate that keratocytes endocytose latex beads and store them for a long time, isolating these foreign materials from the corneal stroma. These observations suggest that keratocytes, like some other fibroblasts perform a noninflammatory and nonimmunological defense function.

Previous in vivo studies evaluating the effects of growth factors on epithelial regeneration have used the scrape injury model in rabbit eyes. Since growth factors act principally on the mitotic activity of regenerating cells, the rapid wound closure rates following scrape injury may not adequately access the effects of these agents on epithelial repair. In this study, we evaluated the rates of wound healing following scrape (8.6 mm) and lamellar keratectomy (8.6 mm) injury in 25 albino rabbits. Eyes were left untreated or received daily application (two to three times) of (a) Tears Naturale II, (b) 50 mM Tris/NaCl, and (c) commercial vehicle for EGF. Eyes were evaluated daily by fluorescein staining with Ophthalmic Fluoro-Strips followed by clinical photography. The area of staining was quantitated by computer-assisted planimetry and rates calculated by linear regression analysis. Eyes receiving scrape injuries epithelialized by 3 days following surgery. Rates of wound closure in two separate groups (six eyes each) were 25.83 mm2/day (r = 0.96) and 29.56 mm2/day 9r = 0.97). Lamellar keratectomy injuries epithelialized by 7 to 8 days, which is substantially longer than that observed for scrape injuries. Rates of wound healing in two separate untreated groups (8 and 10 eyes) were 10.88 mm2/day (r = 0.95) and 9.00 mm2/day (r = 0.93), respectively, which were not significantly different. Analysis of variance comparing rates of wound closure indicated that lamellar keratectomy injuries heal at a significantly slower rate when compared to scrape injury (p less than 0.0005).(ABSTRACT TRUNCATED AT 250 WORDS)

Anterior stromal micropuncture has become an effective treatment for recurrent erosion. The healing process in rabbit corneas was investigated. Following micropuncture of the corneal surface with a 27-gauge needle knife, electron microscopy was carried out at regular intervals from time 0 through 5 months. The corneal incisions began to fill with epithelium by day 1. Activated keratocytes were adjacent to the basement membrane defect by 7 days. The basement membrane appeared to be healed at 2 and 4 weeks. Epithelial projections into the stromal incisions with underlying mature basement membrane persisted at 5 months postsurgery. Basement membrane reproduction occurred much more rapidly following needle puncture than after microdiathermy. This was thought to occur because the corneal epithelial cell was immediately exposed to type I collagen, whereas following microdiathermy, new type I collagen must be secreted on the necrotic collagen before the corneal epithelium will secrete basement membrane.