Biomechanical efficacy of corneal cross-linking using hypoosmolar riboflavin solution.

PURPOSE: The use of hypoosmolar riboflavin solution has been suggested for cross-linking thin corneas. The aim of this study was to compare the biomechanical efficacy of corneal cross-linking using hypoosmolar dextran-free riboflavin solution (HCXL) versus isoosmolar standard corneal cross-linking treatment (CXL). METHODS: A total of 24 postmortem porcine eyes with debrided corneas were subdivided into three treatment groups: Controls, the isoosmolar group with isoosmolar 0.1% riboflavin-20% dextran solution and the hypoosmolar group with dextran-free, 0.1% riboflavin solution. The samples were irradiated with UVA light of 365 nm wavelength and an irradiance of 3 mW/cm² for 30 min (dose 5.4 J/cm²). For the biomechanical measurements, 400-µm-deep anterior corneal flaps were created using a lamellar rotating microkeratome. Uniaxial stress-strain measurements were performed. RESULTS: In the isoosmolar treatment group, stress and Young's modulus at 8% strain were significantly increased by 67.97%, respectively, 62.62% versus the controls. In the hypoosmolar treatment group, stress and Young's modulus at 8% strain were significantly increased by 81.21%, respectively, 51.40% versus the controls. There was no significant difference between the iso- and hypoosmolar groups in biomechanical efficacy. On histology, there was no edema in the anterior 200 µm of the corneas after stromal swelling by the hypoosmolar riboflavin solution. CONCLUSION: Corneal cross-linking using isoosmolar or hypoosmolar riboflavin solution induces a comparable biomechanical effect. This is explained by the localization of the maximum cross-linking effect in the anterior 200 µm of the cornea which are not affected by the swelling effect of hypoosmolar riboflavin solution.

Biomechanical efficacy of contact lens-assisted collagen cross-linking in porcine eyes.

PURPOSE: Contact lens-assisted corneal cross-linking (CACXL) has been proposed for the cross-linking treatment in thin corneas. The aim of this study was to assess the biomechanical efficacy of this treatment. METHODS: Post-mortem porcine eyes were treated with standard cross-linking and with cross-linking placing a contact lens soaked with isoosmolar riboflavin solution on the debrided cornea with or without an adherent precorneal riboflavin film of up to 100 µm thickness. Three soft contact lenses (Air Optix Aqua, SofLens and Galifa) with different degrees of hydrophilic properties were tested. After cross-linking with a surface UVA irradiance of 3 mW/cm² for 30 min (fluence 5.4 J/cm²), a 400 µm deep anterior corneal flap was created using a lamellar rotating microkeratome. Biomechanical stress-strain measurements and thermal shrinkage tests were performed. RESULTS: In the Air Optix Aqua group (30% hydration) without riboflavin film, Young's modulus and stress at 8% strain were increased significantly versus untreated controls and the effect was 92.4% respectively 86.35% of the standard CXL value. In the SofLens group (59% hydration) without riboflavin film, Young's modulus and stress at 8% strain were increased significantly versus untreated controls and the effect was 67.04% respectively 65.28% of the standard CXL value. In the Galifa group (72% hydration) without riboflavin film, Young's modulus and stress at 8% strain were increased significantly versus untreated controls and the effect was about 68.48% respectively 75.52% of the standard CXL value. In all samples with a precorneal riboflavin film under the contact lens, there was no significant biomechanical effect compared to the untreated controls. Similarly, in the hydrothermal experiments at 70°C, there was a typical mushroom pattern with increased resistance to thermal shrinkage in the anterior stroma after standard CXL, a markedly reduced mushroom effect using a riboflavin-soaked contact lens only and no effect with the use of a riboflavin-soaked contact lens plus a precorneal riboflavin film. CONCLUSION: The biomechanical effect of CACXL in porcine corneas is about one-third less than after standard CXL. The efficacy of CACXL might be improved by reducing or omitting the riboflavin film on the contact lens. Further risk assessment studies are necessary.

Biomechanical efficacy of collagen crosslinking in porcine cornea using a femtosecond laser pocket.

PURPOSE: The aim of this study was to assess the biomechanical efficacy of transepithelial collagen crosslinking using the femtosecond laser pocket technique compared with that using the standard crosslinking (CXL) technique. METHODS: Forty ex vivo porcine eyes were divided into 4 groups with 10 samples each. Group 1 comprised the untreated controls. Group 2 was the standard CXL group with debridement, instillation of 0.1% riboflavin-dextran solution for 15 minutes before and every 5 minutes during the 30 minutes of irradiation with ultraviolet A (UVA) light of 370 nm and an irradiance of 3 mW/cm². Group 3 pertained to the femtolaser pocket control with an intrastromal pocket but without riboflavin/UVA. Group 4 was the femtolaser pocket CXL group with an intrastromal pocket of an 8-mm diameter at a 180-µm depth, riboflavin/dextran application for 15 minutes and subsequent exposure to UVA light for 30 minutes. Postoperatively, biomechanical stress-strain measurements were performed. RESULTS: In the standard CXL group, the stress at 10% strain was 207.8 ± 64.1 × 10 Pa (+79.45% vs. controls; P = 0.021) compared with 115.8 ± 20.8 × 10 Pa in the untreated control group; in the crosslinked femtolaser pocket group, it was 159.5 ± 30.4 × 10 Pa (+37.74%; P = 0.049), in the non-cross-linked femtolaser pocket group, it was 103.5 ± 17.3 × 10 Pa (-10.62%; P = 0.103). The Young modulus was 5.4 MPa (+100% vs. controls) in the standard CXL group, 3.7 MPa (+37.04%) in the crosslinked femtolaser pocket group, and 2.4 MPa (-11.12%) in the non-cross-linked femtolaser pocket group compared with 2.7 MPa in the untreated control group. CONCLUSIONS: The biomechanical effect of CXL using the femtolaser pocket technique is about 50% less pronounced than that after standard CXL. Future studies will show whether the efficacy of the technique can still be improved and whether the clinical effect is sufficient for stabilizing ectatic corneas.

Expression analysis of ADAM17 during mouse eye development.

ADAM17 (a disintegrin and metallopeptidase domain 17) is crucial for eye morphogenesis. In this study we analysed the expression pattern of ADAM17 during mouse eye development. ADAM17 expression in adult retina was examined using the reverse transcription-polymerase chain reaction (RT-PCR) and verification of the RT-PCR products by DNA sequencing. Immunohistochemistry was performed to evaluate the ADAM17 expression pattern in mouse eyes at developmental stages of embryonic day (E) 12, E14, E16, E18, postnatal day (P) 0, P1, P4, P7, P14, P 30 and P175 (adult). We detected ADAM17 mRNA in adult retina tissue. ADAM17 protein was expressed in non-pigmented ciliary epithelial cells and in retinal vessels from P7 onwards during eye development. In corneal epithelial cells and endothelium, ADAM17 protein was present from P14 onwards. Although, mice in which the functional ADAM17 gene is significantly reduced develop multiple eye malformations, the expression of ADAM17 is not ubiquitous over the entire eye. Its expression pattern during development suggests that not only TNF-alpha but additional membrane-anchored substrates of ADAM17 play an important role in eye formation.

Limbal and conjunctival epithelium after corneal cross-linking using riboflavin and UVA.

PURPOSE: To assess possible cellular damage in the corneal and conjunctival epithelium after corneal cross-linking treatment. METHODS: Riboflavin-dextran solution was applied every 5 minutes to the right eyes of 3 rabbits 10 minutes before and 30 minutes during the irradiation with UVA light of 370 nm and an irradiance of 3 mW/cm at the 8- to 10-o'clock position, including conjunctival, limbal, and central corneal epithelium. In addition, 3 rabbits were treated with UVA light only. The rabbits were killed 24 hours later. The treated eyes were examined histologically using hematoxylin-eosin and periodic acid-Schiff staining, immunohistochemistry, and a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) apoptosis assay and compared with the left fellow eyes, which served as controls. RESULTS: There was no epithelial defect on fluorescein staining. No apoptosis was found in the corneal limbal epithelium, keratocytes, or endothelial cells in the irradiated area. In the adjacent conjunctival epithelium, rare superficial conjunctival epithelial cells were positive in TUNEL staining in all animals. Anti-multicytokeratin-positive staining was demonstrated in both the limbal corneal and the conjunctival epithelium. The proliferation marker Ki-67 was identified in the basal cell layer of the limbal epithelium. The frequency and distribution pattern of goblet cells, multicytokeratin, and the proliferation marker Ki-67 were the same in all eyes compared with the untreated fellow control eyes. CONCLUSIONS: Standard corneal cross-linking does not induce significant cellular epithelial damage as assessed by histological methods. Further studies on possible genotoxic and long-term changes would be helpful to complete the risk assessment.

Thermomechanical stability of sclera after glyceraldehyde crosslinking.

PURPOSE: Recently, glyceraldehyde-induced crosslinking was proposed by us for the treatment of progressive myopia, increasing significantly the biomechanical rigidity of sclera. The aim of the present study was to investigate the changes in thermo-mechanical stability after scleral glyceraldehyde crosslinking, allowing a better evaluation of the efficacy of crosslinking. METHODS: One hundred and twenty six porcine eyes were retrieved from the local abattoir. Using hot saline solution, the threshold shrinkage temperature (Ts) was determined for both equatorial scleral strips and whole eye globes incubated with glyceraldehyde for 4 days. Untreated control samples and specimens crosslinked with formaldehyde for 4 days were tested for comparison. In the globes, a small 6 mm limbus-parallel scleral strip was excised 5 mm behind the limbus to allow extrusion of the vitreous, facilitating heat-induced globe contraction. After heat exposure, the eyes were examined histologically by light microscopy. RESULTS: There was significant Maillard browning of the sclera after incubation with glyceraldehyde. The contraction temperature determined in the glyceraldehyde group was 78°C for both scleral strips and globes, in the formaldehyde group 88°C for scleral strips and 92°C for globes, and in non-crosslinked controls 62°C for scleral strips and 68°C for globes. Interestingly, the eye balls contracted in an implosion-like manner, leading to an abrupt reduction in eye volume by about one third. On light microscopy, scleral thickening, heat denaturation of collagen fibers, and loss of birefringence were noted. CONCLUSIONS: Scleral collagen crosslinking by glyceraldehyde proved very efficient in increasing the scleral thermomechanical stability by at least 10°C in Ts, stabilizing the eye shape and preventing the shrinkage of the eye in all dimensions. There is hope that, in a similar manner, glyceraldehyde crosslinking can stabilize the scleral collagen crosslinks and eye shape in myopia, stopping progression of scleral thinning and stretching.

Significance of the lacunar hydration pattern after corneal cross linking.

PURPOSE: The aim of the present study was to investigate the characteristic honeycomb hydration pattern after corneal cross linking using in vivo rabbit cornea. METHODS: After removal of the central epithelium, the right corneas of 4 New Zealand white rabbits were cross-linked applying a photosensitizing 0.1% riboflavin-dextran solution and UV-A light of 370 nm wavelength with a surface irradiance of 3 mW/cm for 30 minutes. Two animals were euthanized 3 days postoperatively and another 2 were euthanized 6 weeks postoperatively. The corneas of the enucleated eyes were evaluated using 4-mum light microscopic sections with tangential en face and cross-sectional orientation. RESULTS: By day 3 after treatment, complete apoptotic damage and loss of the stromal keratocytes and endothelial cells were found in the central irradiated area through the entire thickness of the stroma. There was marked lacunar edema in the former positions of the apoptotic keratocytes in the anterior 250 microm of the stroma and diffuse edema in the adjacent posterior and lateral zones. Lacunar edema was identified best on tangential sections. By week 6, the cytoarchitecture of the cornea appeared normal again, and complete resolution of both lacunar and diffuse corneal edema had occurred. CONCLUSIONS: After riboflavin/UV-A cross linking of in vivo rabbit cornea, a characteristic lacunar hydration pattern can be observed in the anterior stroma with maximum cross linking, whereas diffuse edema is present in the adjacent areas without significant cross linking. The lacunar edema may explain the temporary demarcation of the anterior stroma after cross linking on biomicroscopy because of increased light scattering. The network pattern of cross linking may contribute to the elasticity of the cornea after cross linking.

Significance of the riboflavin film in corneal collagen crosslinking.

PURPOSE: To evaluate the role of the preocular riboflavin film in ultraviolet-A (UVA) absorption in corneal collagen crosslinking (CXL). SETTING: Eye Laser Institute, Department of Ophthalmology, Martin-Luther-University, Halle, Germany. METHODS: The absorption of UVA light was measured in human donor and porcine postmortem corneas with and without riboflavin film using 3 solutions: standard dextran-riboflavin, methylcellulose-riboflavin, and hypoosmolar riboflavin-sodium chloride without dextran. The breakup time of the solutions and their absorbance were also determined. RESULTS: After 30-minute instillation of riboflavin solution, the corneal absorption coefficient of the combined stroma-riboflavin film system was 56.36 cm(-1) in human corneas and 51.46 cm(-1) in porcine corneas using dextran-riboflavin; 69.87 cm(-1) and 53.86 cm(-1), respectively, using methylcellulose-riboflavin; and 48.19 cm(-1) and 42.68 cm(-1), respectively, using hypoosmolar riboflavin. For the stroma alone without riboflavin film, the absorption coefficient was reduced to 36.95 cm(-1) in human corneas and 28.91 cm(-1) in porcine corneas using dextran-riboflavin; 38.26 cm(-1) and 32.49 cm(-1), respectively, using methylcellulose-riboflavin; and 38.88 cm(-1) and 28.42 cm(-1), respectively, using hypoosmolar riboflavin solution. The breakup time was 22 minutes for the dextran-riboflavin film, 32 minutes for methylcellulose, and 90 seconds for the hypoosmolar solution. CONCLUSION: Results indicate that the cornea including the riboflavin film can be considered a composite 2-compartment system and that the riboflavin film is an integral part of the CXL procedure and important in achieving the correct stromal and endothelial UVA irradiance. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement.

PURPOSE: To test the biomechanical efficiency of corneal crosslinking with riboflavin without epithelial debridement (C3-R). SETTING: Moscow Helmholtz Research Institute of Eye Diseases, Moscow, Russia. METHODS: The left eyes of rabbits were crosslinked using standard crosslinking including epithelial removal (Group 1), using benzalkonium chloride-containing proxymetacaine eyedrops without epithelial removal (Group 2), or using preservative-free oxybuprocaine eyedrops without epithelial removal (Group 3). All left eyes received riboflavin solution and were irradiated with an ultraviolet-A double diode for 30 minutes (irradiance 3 mW/cm(2)). The animals were killed 1 day after crosslinking. Biomechanical and histological analyses were performed. RESULTS: Fourteen eyes were evaluated. There was a statistically significant increase in Young's modulus in Group 1 (102.45%) and in Group 2 (21.30%). In Group 3, no biomechanical changes were measured. Histology showed complete cell loss of keratocytes and endothelium in Group 1 and inhomogeneous keratocyte loss down to 200.0 microm in Group 2. No changes were observed in Group 3. CONCLUSIONS: Corneal crosslinking without epithelial debridement reduced the biomechanical effect by approximately one fifth compared with standard crosslinking, probably because of restricted and inhomogeneous stromal distribution of riboflavin. The cytotoxic damage was restricted to 200.0 microm stromal depth, which is an advantage over the standard method. Therefore, C3-R is not recommended for the routine treatment of keratoconus but primarily for cases with a corneal thickness less than 400.0 microm in which standard crosslinking cannot be used without serious risk to the endothelium.

Potential use of riboflavin/UVA cross-linking in bullous keratopathy.

BACKGROUND: Collagen cross-linking of the cornea has been shown by us to have an antiedematous effect in the cornea. The aim of the present study was to examine if this effect can be used for the treatment of bullous keratopathy. METHODS: This clinical interventional case series included 3 patients (3 eyes) with bullous keratopathy due to pseudophakia, corneal transplant rejection, and Fuchs' endothelial dystrophy. After dehydration for 1 day using 40% glucose, the central 8 mm of the cornea were abraded and cross-linked with the photosensitizer riboflavin and UVA (370 nm, 3 mW/cm(2)) for 30 min. Optical coherence tomography pachymetry measurements of the central cornea were performed at various time intervals. RESULTS: Corneal thickness was reduced by 90.33 +/- 17.04 microm on average 3 days after cross-linking and by 93.67 +/- 14.22 microm after 8 months. The bullous changes of the epithelium were markedly improved, resulting in loss of pain and discomfort. Visual acuity was significantly improved in the case without prior stromal scarring. CONCLUSIONS: Cross-linking might become another useful tool in the treatment of bullous keratopathy. It is primarily suited for patients with pain symptoms, restricted visual prognosis or to extend the time interval for an upcoming corneal transplantation.

Long-term biomechanical properties of rabbit cornea after photodynamic collagen crosslinking.

PURPOSE: Photodynamic riboflavin/ultraviolet-A (UVA)-induced collagen cross-linking, which increases the biomechanical stiffness of the human cornea by about 300%, has been introduced recently as a possible treatment for progressive keratoconus. The present study was undertaken to evaluate the longterm biomechanical effects of this new cross-linking treatment as a necessary prerequisite to its clinical success. METHODS: The corneas of the left eyes of nine male rabbits were cross-linked. The contralateral eyes served as controls. After removal of the central 7 mm of the epithelium, the corneas were treated with the photosensitizer riboflavin and UVA irradiation for 30 mins with an irradiance of 3 mW/cm(2) using a 370-nm UVA double diode. Groups of three animals were killed immediately after treatment and at 3 and 8 months, respectively. Biomechanical stress-strain measurements were performed using a microcomputer-controlled biomaterial tester on 4 x 10-mm corneal strips. RESULTS: Corneal thickness in the treated rabbit cornea was 408 +/- 20 microm. A constant and significant increase in ultimate stress (of 69.7-106.0%), Young's modulus of elasticity (of 78.4-87.4%) and a decrease in ultimate strain (of 0.57-78.4%) were found over a time period of up to 8 months after cross-linking treatment. CONCLUSIONS: Riboflavin/UVA-induced collagen cross-linking leads to a longterm increase in biomechanical rigidity which remains stable over time. These data support our previous longterm clinical observations and give hope that this new treatment will halt progressive keratoconus definitively.

Long-term biomechanical properties of rabbit sclera after collagen crosslinking using riboflavin and ultraviolet A (UVA).

PURPOSE: Scleral crosslinking by the photosensitizer riboflavin and ultraviolet A (UVA) has been shown to increase significantly the scleral biomechanical rigidity and might therefore become a possible sclera-based treatment modality for progressive myopia. In the present study, the long-term effect of the new crosslinking method on biomechanical properties was investigated in the rabbit sclera. METHODS: A 10 x 10 mm sector of the equatorial sclera of nine Chinchilla rabbit eyes was treated in vivo using a UVA double diode of 370 nm with a surface irradiance of 3 mW/cm(2) and application of 0.1% riboflavin-5-phosphate drops as photosensitizer for 30 min. Three days, 4 months and 8 months postoperatively, biomechanical stress-strain measurements of the treated scleral strips were performed and compared to contralateral control sclera using a microcomputer-controlled biomaterial tester. In addition, routine histological controls were performed. RESULTS: Following the crosslinking treatment, Young's modulus was increased by 320% after 3 days, 277% after 4 months and 502% after 8 months, and ultimate stress by 341% after 3 days, 131% after 4 months and 213.8% after 8 months versus the controls. The decrease in ultimate strain was between 24% and 44.8%. On histology, no tissue damage was detected. CONCLUSION: Our new method of scleral collagen crosslinking proved very effective and constant over a time interval of up to 8 months in increasing the scleral biomechanical strength. Therefore, the new treatment might become an option for strengthening scleral tissue in progressive myopia and other conditions associated with weakened sclera. There were no side-effects on the retina or retinal pigment epithelium. The new crosslinking treatment could now be tested in a suitable myopia model (like the tree shrew) and finally in human eyes.

Long-term biomechanical properties after collagen crosslinking of sclera using glyceraldehyde.

PURPOSE: Chemical crosslinking by glyceraldehyde has been shown to increase significantly the biomechanical rigidity of sclera. It might therefore become an option for a sclera-based treatment of progressive myopia. The present pilot study was designed to test the long-term biomechanical efficiency of the new crosslinking method. METHODS: Six Chinchilla rabbits were treated with sequential sub-Tenon's injections of 0.15 ml 0.5 m glyceraldehyde, which were given in the supero-nasal quadrant of the right eye (OD) five times over 14 days. The rabbits were killed 4 months and 8 months after crosslinking treatment, respectively. Biomechanical stress-strain measurements of scleral strips from the treatment area were performed and compared to non-treated contralateral control sclera using a microcomputer-controlled biomaterial testing device. In addition, the eyes were examined histologically by light microscopy to evaluate possible side-effects. RESULTS: Following the crosslinking treatment, the ultimate stress was 10.2 +/- 2.3 MPa after 4 months and 8.5 +/- 2.2 MPa after 8 months versus 2.4 +/- 0.3 MPa in the controls (increases of 325% and 254.17%, respectively); Young's modulus was 104.6 +/- 13.7 MPa after 4 months and 53.2 +/- 5.2 MPa after 8 months versus 9.6 +/- 1.3 MPa in the controls (increases of 989.6% and 554.17%, respectively); and ultimate strain was 15.8 +/- 1.5% after 4 months and 24.1 +/- 0.7% after 8 months versus 38.4 +/- 4.6% in the controls (decreases of 58.84% and 37.24%, respectively). Histologically, no side-effects were found. CONCLUSION: Our new method of scleral collagen crosslinking proved very efficient in increasing scleral biomechanical strength over a period of up to 8 months. Glyceraldehyde can be applied easily by sequential parabulbar injections. Before clinical application in myopic patients, a study in an animal myopia model is recommended.

Biomechanical changes of the internal limiting membrane after indocyanine green staining.

Selective indocyanine green (ICG) staining of the macula has recently become popular in internal limiting membrane (ILM) peeling allowing a better distinction of the ILM from the underlying retina. Clinically, the ILM seems to become stiffer after ICG staining facilitating ILM peeling for the retinal surgeon. In the present study, we tried to verify the cause of this biomechanical effect. Retinal samples of postmortem porcine eyes were treated with ICG and light and compared to samples treated in darkness using biomechanical force and elongation measurements. After ICG staining of the retina combined with a 3-min illumination, a significant increase in ultimate force by 45% and a decrease in ultimate elongation by 24% were found indicating greater stiffness of the ICG-stained ILM. Without light exposure there was no such effect suggesting a light-dependent process. The stiffening effect of ICG and light is due to a photosensitizing effect of ICG leading to collagen cross-linking of the ILM.

Gel electrophoretic analysis of corneal collagen after photodynamic cross-linking treatment.

PURPOSE: Photodynamic collagen cross-linking by using ultraviolet A (UVA) irradiation and the photosensitizer riboflavin has been recently introduced as a new possible treatment of progressive keratoconus. This is the first study, to our knowledge, investigating biochemical aspects of the new procedure. Its aim was to analyze the possible changes in the electrophoretic pattern of corneal collagen type I after collagen cross-linking treatment. METHODS: Twenty fresh postmortem porcine corneas were cross-linked; another 20 porcine corneas treated with physiologic saline were used as controls. After removal of the central 10 mm of the epithelium, the corneas were treated with the photosensitizer riboflavin and UVA irradiation for 30 minutes by using a double UVA diode (370 nm, 3 mW/cm). For biochemical analysis, the central 10-mm corneal buttons were trephined, tissue was homogenized, and collagen type I was extracted. Subsequently, the collagen extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS: In the controls, the typical collagen pattern of normal cornea was found with 1 gamma trimer band, 2 beta dimer bands, and 2 alpha monomer bands. In the cross-linked samples, there was an additional intense polymer band in the stacking gel that was resistant to mercaptoethanol, heat, and pepsin treatment. Its molecular size was estimated to be at least 1000 kDa. CONCLUSIONS: In the cross-linked corneas, a strong band of high-molecular-weight collagen polymers was shown as the biochemical correlate of the cross-linking effect, showing the efficiency of the new cross-linking procedure. This polymer band complies well with the morphologic correlate of an increased fiber diameter after cross-linking treatment. Its chemical stability supports hopes of a long-term effect of the new treatment.

Crosslinking of scleral collagen in the rabbit using glyceraldehyde.

PURPOSE: To strengthen rabbit sclera in vivo using chemical crosslinking with glyceraldehyde for a scleral-based treatment of progressive myopia. SETTING: Department of Ophthalmology, Martin-Luther-University, Halle, Germany. METHODS: Five chinchilla rabbits were treated with sequential sub-Tenon injections of 0.15 mL 0.5 M glyceraldehyde into the superonasal quadrant of the right eye 5 times during 14 days. The rabbits were humanely killed and biomechanical stress-strain measurements of scleral strips from the treatment area were performed and compared with nontreated contralateral control sclera using a microcomputer-controlled biomaterial tester. The treated eyes were examined histologically by light microscopy to exclude possible adverse effects. RESULTS: Following the crosslinking treatment, the ultimate stress was 15.8 MPa +/- 6.0 (SD) versus 3.1 +/- 0.3 MPa in the controls (increase of 409.7%; P<.02), the Young modulus was 129.6 +/- 53.7 MPa versus 11.5 +/- 1.8 MPa in the controls (increase of 1027%, P<.01), and ultimate strain was 19.8% +/- 2.6% MPA versus 38.2% +/- 5.1% MPA in the controls (decrease of 48.2% P<.05). Histologically, mild side effects were found in the peripheral cornea adjacent to the treatment area, with some inflammatory infiltrate and moderate loss of keratocytes. CONCLUSIONS: Glyceraldehyde crosslinking of scleral collagen increased the scleral biomechanical rigidity efficiently. Glyceraldehyde can be easily applied by sequential parabulbar injections. There were no side effects on the retina, so the new method might become a treatment modality for strengthening scleral tissue to prevent progressive myopia.