Corneal metabolic biomarkers for moderate and high myopia in human.

This study aimed to identify the corneal metabolic biomarkers for moderate and high myopia in human. We enrolled 221 eyes from 221 subjects with myopia to perform the femtosecond laser small incision lenticule extraction (SMILE) surgery. Among these, 71 eyes of 71 subjects were enrolled in the low myopic group, 75 eyes of 75 subjects in the moderate myopic group and 75 eyes of 75 subjects in the high myopic group. The untargeted metabolomics analysis was performed to analyze the corneal tissues extracted during the SMILE surgery using an ultra-high-performance liquid chromatography (UHPLC) coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometry (MS). The one-way analysis of variance (ANOVA) was used to identify the different metabolites among the three myopic groups, the orthogonal partial least-squares discriminant analysis (OPLS-DA) model was used to reveal the different metabolites between moderate myopia and low myopia, and between high myopia and low myopia. The Venn gram was used to find the overlapped metabolites of the three datasets of the different metabolites. The stepwise multiple linear regression analysis was used to determine the metabolic molecules associated with manifest refractive spherical equivalents (MRSE). The Receiver Operating Characteristics (ROC) analysis was performed to reveal the corneal biomarkers for moderate and high myopia. The hub biomarker was further selected by the networks among different metabolites created by the Cytoscape software. A total of 1594 metabolites were identified in myopic corneas. 321 metabolites were different among the three myopic groups, 106 metabolites were different between high myopic corneas and low myopic corneas, 104 metabolites were different between moderate myopic corneas and low myopic corneas, and 30 metabolic molecules overlapped among the three datasets. The multivariate linear regression analysis revealed the myopic degree was significantly influenced by the corneal levels of azelaic acid, arginine-proline (Arg-Pro), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, and hypoxanthine. The ROC curve analysis showed that azelaic acid, Arg-Pro and hypoxanthine were effective in discriminating low myopia from moderate to high myopia with the area under the curve (AUC) values as 0.982, 0.991 and 0.982 for azelaic acid, Arg-Pro and hypoxanthine respectively. The network analysis suggested that Arg-Pro had the maximum connections among these three biomarkers. Thus, this study identified azelaic acid, Arg-Pro and hypoxanthine as corneal biomarkers to discriminate low myopia from moderate to high myopia, with Arg-Pro serving as the hub biomarker for moderate and high myopia.

Ocular safety evaluation of blue light scleral cross-linking in vivo in rhesus macaques.

PURPOSE: To investigate the safety of blue light scleral cross-linking (SXL) by evaluating changes in biological parameters in the retina and choroid in the eyes of rhesus macaques (Macaca mulatta). METHODS: Fifteen 3-year-old macaques (30 eyes) were randomly divided into three groups (n = 5). SXL was performed via riboflavin (0.5%) and blue light (460 nm) at the location of the equatorial sclera. Right eyes served as experimental eyes, and left eyes as control eyes. One quadrant of each right eye was irradiated in group A, two quadrants of each right eye and one quadrant of each left eye were irradiated in group B, and two quadrants of each right eye were irradiated in group C. Optical coherence tomography, optical coherence tomography angiography, and flash electroretinography (f-ERG) examinations were performed at baseline and 1 week, 1 month, 3 months, and 6 months after SXL. Additionally, retinal tissue alterations were detected via transmission electron microscopy at 1 week postoperatively. RESULTS: There were no significant differences between experimental eyes and control eyes in retinal thickness, vessel density of retinal superficial capillary plexus, and choroid thickness in any of the groups at any of the time points investigated (p > 0.05). Significant reductions in f-ERG parameters were detected 1 week postoperatively in the experimental eyes of groups A and C (p < 0.05), but they gradually recovered, and there was no significant difference 1 month postoperatively (p > 0.05). Ultrastructural changes were evident in the retinal layers of SXL eyes. In group B, there were no significant differences between the right and left eyes at any of the follow-up time points investigated. CONCLUSIONS: Blue light SXL can cause transient retina damage. The f-ERG parameters reductions and retinal ultrastructural changes were found at early stage, even though there were not significant changes in retinal thickness, vessel density of retinal superficial capillary plexus, and choroid thickness after blue light SXL. The long-term intraocular safety of the blue light SXL technique should be investigated further.

Clinical Feasibility and Safety of Scleral Collagen Cross-Linking by Riboflavin and Ultraviolet A in Pathological Myopia Blindness: A Pilot Study.

INTRODUCTION: To investigate the feasibility and safety of scleral ultraviolet A (UVA) cross-linking (scleral CXL) on pathologically blindness. METHODS: This was a prospective, observational clinical study. Five patients with monocular blindness due to pathological myopic maculopathy were enrolled. Eyes with best corrected visual acuity (BCVA) under 0.05 were defined as experimental eyes. The fellow eyes were defined as control eyes. Patients first underwent posterior scleral reinforcement (PSR) surgery in the control eye. Thereafter, scleral CXL surgery was performed in the experimental eye on the same day. Visual acuity, BCVA, slit lamp biomicroscopic examination, intraocular pressure measurement, corneal specula microscopies, axis length measurement, funduscopy with pupil dilation, color fundus photography, full-field flash electroretinography, optical coherence tomography, and color Doppler flow imaging were performed at baseline, 1 week, 1 month, 3 months, 6 months, and 12 months after surgery. RESULTS: No signs of inflammation were observed after operation and throughout the follow-up period. Retinoschisis was improved, while choroidal neovascularization fibrosis and retinal and choroidal atrophy were unchanged after scleral CXL. There were no statistically significant differences in the ophthalmic artery, central retinal artery, and posterior ciliary artery parameters of color Doppler flow imaging or in retinal thickness, within experimental and control eyes, at baseline, 1 week, 1 month, 3 months, or 12 months (P > 0.05). CONCLUSIONS: This pilot study verified the feasibility and safety of scleral CXL on human blindness. The UVA-CXL on the sclera of human eyes seems to have the same effect as PSR in preventing progressive pathological myopia in the future. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR2100042422).

Effects of riboflavin/ultraviolet-A scleral collagen cross-linking on regional scleral thickness and expression of MMP-2 and MT1-MMP in myopic guinea pigs.

OBJECTIVE: To investigate the effects of scleral collagen cross-linking (SXL) using riboflavin and ultraviolet A (UVA) light on the scleral thickness of different regions and expression of matrix metalloproteinase 2 (MMP-2) and membrane-type MMP-1 (MT1-MMP) in guinea pigs with lens-induced myopia. METHODS: Forty-eight 4-week-old guinea pigs were assigned to three groups (n = 16 per group): SXL group, lens-induced myopia (LIM) group, and control group. The sclera of the right eye of the guinea pig in the SXL group was surgically exposed, riboflavin was dropped on the treatment area for 10 minutes before the 30-minute UVA irradiation. The same surgical procedure was performed in the LIM group without UVA irradiation. The -10.00 D lenses were then placed on the right eyes of guinea pigs in the SXL and LIM groups for six weeks. The control group received no treatment. The left eyes were untreated in all groups. The ocular axial length (AXL) and refraction were measured at 4 weeks and 10 weeks of age. 10-week-old guinea pigs were sacrificed, and the right eyes were enucleated and evenly divided for preparation of hematoxylin and eosin (HE) stained sections, quantitative real-time polymerase chain reaction (qPCR) and western blotting. The scleral thickness of different regions was measured on HE stained sections. The temporal half of the sclera was harvested to measure the expression of MMP-2 and MT1-MMP by qPCR and western blotting. RESULTS: The AXL was significantly shorter, and the degree of myopic refraction was significantly lower in the SXL group than those in the LIM group at 10 weeks of age. The scleral thickness of the cross-linked area was significantly greater in the SXL group than that of the corresponding area in the LIM group, while the scleral thickness of the untreated nasal side was not significantly different between the SXL group and the LIM group. The expression of MMP-2 and MT1-MMP of the cross-linked sclera was significantly downregulated compared with that of the corresponding area in the LIM group. CONCLUSION: Riboflavin/UVA SXL could slow myopia progression and thicken the cross-linked sclera in guinea pigs, which might be related to the downregulation of MMP-2 and MT1-MMP expression during the scleral remodeling process.

Scleral Remolding-Related Gene Expression After Scleral Collagen Cross-Linking Using Ultraviolet A and Riboflavin in Myopic Guinea Pig Model.

PURPOSE: This study was conducted to evaluate scleral remolding-related gene expression after scleral collagen cross-linking (SCXL) using ultraviolet A (UVA) and riboflavin in lens-induced myopia (LIM) guinea pigs. METHODS: A total of 100 4-week-old pigmented guinea pigs were randomly divided into five groups (n = 20): SCXL + LIM, LIM, SCXL, Sham, and Control. Refraction, anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), and axial length (AL) were measured using streak retinoscope and A-scan ultrasonography. SCXL was performed using 0.1% riboflavin solution and 365 nm UVA irradiation. Lens-induced myopia was achieved by wearing -10 D concave lenses. Quantitative real-time PCR (qPCR) and western blot were used to measure mRNA and protein levels, respectively. RESULTS: Myopia was successfully induced in the LIM group, while myopic refraction was higher and ACD and AL were shorter in SCXL + LIM compared with LIM, suppressing myopia progression. The scleral COL1A1 mRNA levels were significantly decreased and MMP2 and ACTA2 mRNA levels were significantly increased in LIM compared with other groups, while COL1A1 mRNA levels were increased and MMP2 and ACTA2 mRNA levels were decreased in SCXL + LIM compared with LIM. The scleral COL1A1 protein levels were significantly increased at 1 week and 4 weeks and MMP2 protein levels were significantly decreased at 1 week in SCXL compared with SCXL + LIM, LIM and Control. MMP2 protein levels were significantly decreased in SCXL + LIM and SCXL compared with LIM at 4 weeks. The differences in TGFB1, BMP2, CCN2, ITGA2, and ITGB1 mRNA levels and ACTA2 protein levels between the five groups were not significantly different. CONCLUSION: SCXL using UVA and riboflavin could influence the expression of scleral remolding-related genes, including COL1A1, MMP2, TIMP2, and ACTA2, and thus contribute to improving collagen synthesis and reducing collagen degradation and might have an effect on slowing myopia progression.

Comparing the Differences in Slowing Myopia Progression by Riboflavin/Ultraviolet A Scleral Cross-linking before and after Lens-induced Myopia in Guinea Pigs.

PURPOSE: To compare the effectiveness and differences in slowing myopia progression in Guinea pigs by riboflavin/ultraviolet A (UVA) scleral cross-linking (sCXL) before and after lens-induced myopia (LIM). METHODS: Forty 4-week-old Guinea pigs were randomly divided into four groups (n = 10 per group): CXL-A, CXL-B, LIM, and Control groups. The right eyes in CXL-A, CXL-B, LIM groups were treated with -10.00 D lenses from 4 to 10-week old and the left eyes were untreated. In CXL-A and CXL-B groups, riboflavin/UVA sCXL was performed on the right eyes at 4 weeks and 8 weeks of age, respectively. Both eyes were untreated in Control group. The intraocular pressure (IOP), the axial length (AXL), and the refraction were measured in vivo at 4, 8, and 10 weeks of age. At 10 weeks of age, the right eyes were enucleated for the tensile test and transmission electron microscopy observations. RESULTS: The myopia has been successfully induced in LIM and CXL-B groups during 4-8 weeks. In CXL-A group, the growth rate of AXL and myopic refraction was markedly inhibited during 4-8 weeks and the inhibitory effects diminished during 8-10 weeks. During 8-10 weeks, the growth rate of AXL and myopic refraction in CXL-B were marked suppressed. At 10 weeks of age, the myopia refraction was lower and the AXL was shorter in CXL-A group in comparison to CXL-B group. The IOP was not significantly different among the 4 groups of eyes at 4, 8, and 10 weeks of age. The scleral stiffness, the fibril diameters, and the fibril density of the sclera were significantly increased in CXL-A and CXL-B groups compared to LIM group. CONCLUSION: Riboflavin/UVA sCXL administrated before and after the myopia modeling could both slow the myopia progression in Guinea pigs. The before-myopia preventative sCXL showed lower myopic refraction in the same age comparison between the cross-linked groups. The effect of riboflavin/UVA sCXL might reduce over time and the long-term effect should be further investigated. This sCXL intervention might control the ultrastructure alterations of the sclera during the myopia remodeling.

Safety and Long-term Scleral Biomechanical Stability of Rhesus Eyes after Scleral Cross-linking by Blue Light.

Purpose: To assess the safety and long-term scleral biomechanical stability of rhesus eyes after blue light scleral CXL by investigating the biomechanical and microstructural changes.Methods: Seven rhesus monkeys (14 eyes) were observed in this study. All right eyes received blue light scleral CXL at the superior temporal equatorial sclera, and the left eyes served as controls. Biological ocular parameters were followed up to 1 year after scleral CXL. Stress-strain measurements of three rhesus sclera were measured, three rhesus retinas were examined histologically by H&E and TUNEL staining. And the microstructure of both the sclera and retina were observed by transmission electron microscopy at 1 year.Results: As for the retinal thickness, choroidal thickness, flow density of retinal superficial vascular networks and flash electroretinography (f-ERG) results, no significant differences were observed between the paired eyes at 1 year (P >.05). At the same time, the scleral collagen fibril distribution was much tighter, and the scleral biomechanical properties were significantly increased in the experimental eyes. However, apoptotic cells and retinal ultrastructural changes could still be found in the retina of the experimental eyes.Conclusion: This study demonstrates that blue light scleral CXL could effectively increase the scleral stiffness of the rhesus eye for at least 1 year, but ultrastructural change was still observed in the retina of scleral CXL eye. Therefore, the long-term intraocular safety of the blue light scleral CXL technique for preventing myopia progression should be investigated further.

Evaluation of the Safety and Long-term Scleral Biomechanical Stability of UVA Cross-linking on Scleral Collagen in Rhesus Monkeys.

PURPOSE: To investigate the changes of retinal and choroidal parameters, scleral biomechanical strength, and ocular histopathology after scleral ultraviolet-A (UVA) cross-linking (CXL) in rhesus monkeys eyes, and to evaluate the safety and long-term biomechanical stability of scleral CXL for preventing myopia from progressing further in clinic. METHODS: Six 3-year-old male rhesus monkeys (12 eyes) were randomized to receive UVA-CXL procedures applied on the superotemporal equatorial sclera. Optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) were used for examination before and 1 week and 1, 3, 6, and 12 months after CXL. The stress-strain behaviors of equatorial scleral strips were analyzed 12 months postoperatively by a biomaterial tester. Hematoxylin-eosin and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining were performed 12 months postoperatively. RESULTS: For central retinal thickness, choroidal thickness, and flow density of central retinal superficial vascular networks, no statistical difference was noted between CXL eyes and control eyes at 12 months postoperatively (P > .05). The biomechanical stability of sclera was increased. The scleral stress and Young modulus at 8% strain corresponded to 184% and 183%, respectively, of the control values at 12 months (each P < .001). No retinal damage was detected on histology in scleral CXL eyes. There was no obvious difference between scleral CXL eyes and control eyes by hematoxylin-eosin and TUNEL staining (P > .05). CONCLUSIONS: Scleral CXL with riboflavin/UVA in rhesus monkey eyes could strengthen the biomechanical properties of scleral tissues and maintain the stability for 12 months postoperatively. The UVA-CXL on the sclera of rhesus monkey eyes seems to be effective and safe. [J Refract Surg. 2020;36(10):696-702.].

Lenticule Thickness Accuracy and Influence in Predictability and Stability for Different Refractive Errors after SMILE in Chinese Myopic Eyes.

Purpose: To evaluate the differences between the estimated and measured lenticule thickness and relationships in myopic eyes with different refractive errors after small-incision lenticule extraction (SMILE). Methods: This prospective study included a total of 190 eyes (96 patients) with a manifest refraction spherical equivalent (MRSE) of -6.16 ± 1.71 D. The measured lenticule thickness was obtained from digital spectral-domain optical coherence tomography image analysis. The estimated lenticule thickness was obtained from the software program. Comparative statistics and linear regression analyses were performed. Results: At 1 month, the estimated lenticule thickness was 113.68 ± 19.35 µm and the measured lenticule thickness was 96.9 ± 17.5 µm. The difference between the estimated and measured lenticule thickness was 13.63 ± 9.1 µm (p < 0.001) thicker. The difference was lower than the mean data in the moderate myopia group (9.7 ± 6.4 µm) and larger in the high myopia group (12.3 ± 8.8 µm) and the super-high myopia group (17.9 ± 6.9 µm). The discrepancy between the estimated and measured lenticule thickness was significantly dependent on the MRSE (p < 0.001). MRSE only showed significant differences between the three and six-month follow-up visit in the super-high myopic group (p = 0.03). Conclusion: The measured lenticule thickness was thinner than the estimated lenticule thickness for SMILE in most eyes. The higher the refraction, the larger the difference after SMILE surgery. The mismatch between the estimated and measured thickness did not influence the refractive precision and stability of different refractive errors.

Early Changes of Ocular Biological Parameters in Rhesus Monkeys After Scleral Cross-linking With Riboflavin/Ultraviolet-A.

PURPOSE: To evaluate the ocular biological parameter difference between scleral corneal cross-linking (CXL) and control eyes in rhesus monkeys by using a rebound tonometer, A-scan ultrasonography, retinoscopy, optical coherence tomography, and electroretinography (ERG). METHODS: Six rhesus monkeys were used in this study, with ages ranging from 3 to 3.5 years. One eye of each rhesus monkey was randomly selected to receive riboflavin/ultraviolet-A CXL in the temporal quadrant of the equatorial sclera and the contralateral eye served as an intra-individual control. The ocular biological parameters were repeatedly measured in both eyes of the monkeys before scleral CXL and 1 week, 1 month, and 3 months postoperatively. RESULTS: The intraocular pressure, refractive state, total axial length, and axial dimensions of the anterior chamber, crystalline lens, vitreous chamber, and central corneal thickness were not statistically significantly different between the control and cross-linked specimens at the different time periods (each P > .05). No obvious changes in the waveform of the standard full-field ERGs were observed in the control and cross-linked specimens. There were no statistically significant differences between the control and cross-linked specimens in the dark-adapted 0.01 ERG, the dark-adapted 3.0 ERG, the light-adapted 3.0 ERG, and the amplitudes of the a-wave and b-wave for the different time periods (each P >.05). CONCLUSIONS: The scleral CXL laboratory technique might not significantly affect the ocular biological parameters of the rhesus monkey in the early postoperative period, but long-term effects and histological changes still need to be investigated further. [J Refract Surg. 2019;35(5):333-339.].

Consistent comparison of angle Kappa adjustment between Oculyzer and Topolyzer Vario topography guided LASIK for myopia by EX500 excimer laser.

AIM: To evaluate and compare the uniformity of angle Kappa adjustment between Oculyzer and Topolyzer Vario topography guided ablation of laser in situ keratomileusis (LASIK) by EX500 excimer laser for myopia. METHODS: Totally 145 cases (290 consecutive eyes )with myopia received LASIK with a target of emmetropia. The ablation for 86 cases (172 eyes) was guided manually based on Oculyzer topography (study group), while the ablation for 59 cases (118 eyes) was guided automatically by Topolyzer Vario topography (control group). Measurement of adjustment values included data respectively in horizontal and vertical direction of cornea. RESULTS: Horizontally, synclastic adjustment between manually actual values (dxmanu) and Oculyzer topography guided data (dxocu) accounts 35.5% in study group, with mean dxmanu/dxocu of 0.78±0.48; while in control group, synclastic adjustment between automatically actual values (dxauto) and Oculyzer topography data (dxocu) accounts 54.2%, with mean dxauto/dxocu of 0.79±0.66. Vertically, synclastic adjustment between dymanu and dyocu accounts 55.2% in study group, with mean dymanu/dyocu of 0.61±0.42; while in control group, synclastic adjustment between dyauto and dyocu accounts 66.1%, with mean dyauto/dyocu of 0.66±0.65. There was no statistically significant difference in ratio of actual values/Oculyzer topography guided data in horizontal and vertical direction between two groups (P=0.951, 0.621). CONCLUSION: There is high consistency in angle Kappa adjustment guided manually by Oculyzer and guided automatically by Topolyzer Vario topography during corneal refractive surgery by WaveLight EX500 excimer laser.

Study of retina and choroid biological parameters of rhesus monkeys eyes on scleral collagen cross-linking by riboflavin and ultraviolet A.

To evaluate ocular fundus biological changes after scleral collagen cross-linking (CXL) with riboflavin/ ultraviolet A (UVA) on rhesus monkeys in vivo by analyzing retina and choroid biological parameters. Six 3-year-old male rhesus monkeys (12 eyes) were observed in this study, with scleral CXL procedures applied on superior temporal equatorial sclera on random eyes of all rhesus. Optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) examination were performed before and 1 week, 1 month, 3 months and 6 months after CXL. The thickness of retina and choroid and the flow density of retinal superficial vascular networks were analyzed respectively in different regions after CXL. As for retina thickness and flow density of retinal superficial vascular networks, no statistical difference was noted between CXL eyes and control eyes at 1 day, 1week, 1 month, 3 months and 6 months (P>0.05). Among choroid parameters, the choroidal thickness in 1500µm temporal to the fovea center of CXL eyes revealed a significant reduction in 1 week postoperatively (P<0.05), but it subsequently increased from 1 month postoperatively, and no statistical difference was found between two groups in the following periods (P>0.05). The choroidal thickness nearby crosslinked region may change temporarily following scleral CXL, and it might recover gradually after 1 month postoperatively. The vascular flow density and thickness of retina were not affected by scleral CXL. Further study should be performed to evaluate the potential adverse effects at the direct vicinity of the application site and the long-term effect of scleral CXL in clinical application.

Prevalence of transforming growth factor ß-induced gene corneal dystrophies in Chinese refractive surgery candidates.

PURPOSE: To determine the prevalence of the transforming growth factor (TGF) ß-induced gene corneal dystrophies in refractive surgery candidates in China. SETTING: Five hospitals in China. DESIGN: Prospective case series. METHOD: Refractive surgical candidates from 5 preselected eye hospitals/centers in China were recruited after providing informed consent. All patients had slitlamp biomicroscopy and collection of a buccal swab as a source of DNA for screening of the TGF ß-induced gene for the 5 most common mutations associated with Reis-Bückler corneal dystrophy, Thiel-Behnke corneal dystrophy, granular corneal dystrophy type 1, granular corneal dystrophy type 2, and lattice corneal dystrophy type 1. RESULTS: Of the 2068 refractive surgery candidates analyzed, 4 had corneal opacities in both eyes on slitlamp examination. Screening for the TGF ß-induced gene found the heterozygous p.R124H mutation associated with granular corneal dystrophy type 2 in each of the 4 individuals with corneal opacities as well as in a fifth individual who did not have any corneal opacities, for a prevalence of 0.24%. Exacerbation of dystrophic corneal deposition developed after laser refractive surgery in 2 individuals who did not have preoperative TGF ß-induced gene screening. CONCLUSIONS: The prevalence of the TGF ß-induced gene corneal dystrophies in Chinese refractive surgery candidates was estimated to be approximately 0.24%. Genetic testing is recommended to identify and exclude from candidacy all individuals with a TGF ß-induced gene dystrophy before elective keratorefractive surgery to avoid causing accelerated postoperative dystrophic deposition.

Effect of a Single Nucleotide Polymorphism in the LAMA1 Promoter Region on Transcriptional Activity: Implication for Pathological Myopia.

PURPOSE: To elucidate the role of the protein coding laminin α1 (LAMA1) gene in pathological myopia (PM) at the transcriptional level. To achieve this, the binding affinity of single nucleotide polymorphism (SNP) rs2089760-located on the LAMA1 promoter gene-to human fetal scleral fibroblast (HFSF) nucleoprotein was investigated and its effect on LAMA1 transcriptional initiation activity was analyzed. METHODS: Binding interactions of the HFSF nucleoprotein and biotin-labeled SNP rs2089760 probe were investigated by amplifying the LAMA1 promoter gene and performing overlap extension polymerase chain reaction (PCR) to obtain the G/A mutation of LAMA1 SNP rs2089760. Recombinant adenovirus vectors, Ad5f11p-pLAMA1SNPa-Luc2, Ad5f11p-pLAMA1SNPg-Luc2, and Ad5f11p-CMV-RLuc, were constructed. Fluorescence intensity ratios of firefly luciferase (FLuc) and renilla luciferase (RLuc) vectors were measured 48 h after HFSF infection. RESULTS: Both specific and mutant probes banded precisely with HFSF nucleoprotein. The intensity value of the mutant probe was significantly lower than that of the specific probe (p < 0.05). HFSFs were successfully infected by the recombinant adenoviruses. The FLuc/RLuc fluorescence intensity ratio of Ad5f11p-pLAMA1SNPa-Luc2 (0.0238 ± 0.0009) was significantly lower than that of Ad5f11p-pLAMA1SNPg-Luc2 (0.0281 ± 0.0015) (p < 0.05). CONCLUSIONS: It is highly likely that SNP rs2089760 in the LAMA1 promoter region is located at the transcription factor binding site. The SNP rs2089760 G > A mutation reduces transcription factor binding ability and transcriptional initiation activity, and negatively regulates gene transcription of LAMA1. We suggest that LAMA1 SNP rs2089760 plays an important role in the development of PM.

Enlargement of the Axial Length and Altered Ultrastructural Features of the Sclera in a Mutant Lumican Transgenic Mouse Model.

Lumican (LUM) is a candidate gene for myopia in the MYP3 locus. In this study, a mutant lumican (L199P) transgenic mouse model was established to investigate the axial length changes and ultrastructural features of the sclera. The mouse model was established by pronuclear microinjection. Transgenic mice and wild-type B6 mice were killed at eight weeks of age. Gene expression levels of LUM and collagen type I (COL1) in the sclera were analyzed by quantitative real-time polymerase chain reaction (qPCR), and the protein levels were assessed by Western blot analysis. Ocular axial lengths were measured on the enucleated whole eye under a dissecting microscope. Ultrastructural features of collagen fibrils in the sclera were examined with transmission electron microscopy (TEM). Lumican and collagen type I were both elevated at the transcriptional and protein levels. The mean axial length of eyes in the transgenic mice was significantly longer than that in the wild-type mice (3,231.0 ± 11.2 µm (transgenic group) vs 3,199.7 ± 11.1 µm (controls), p<0.05 =). Some ultrastructural changes were observed in the sclera of the transgenic mice under TEM, such as evident lamellar disorganizations and abnormal inter-fibril spacing. The average collagen fibril diameter was smaller than that in their wild-type counterparts. These results indicate that the ectopic mutant lumican (L199P) may induce enlargement of axial lengths and abnormal structures and distributions of collagen fibrils in mouse sclera. This transgenic mouse model can be used for the mechanistic study of myopia.