Objective To investigate the feasibility of photochemical tissue bonding (PTB) technique in repairing limbal stem cell (LSC) deficiency and the effect on cornea wound healing. Methods LSCs were isolated from limbus of New Zealand rabbits by tissue block culture method, and then the LSCs of 2nd passage were cultured on de-epithelialized human amniotic membrane (HAM) for 3 weeks to prepare the HAM/LSC grafts. The LSC deficiency models of the left eyes were established by 0.5 mol/L NaOH in 24 New Zealand female rabbits, aged 3-4 months and weighing 1.5-2.0 kg. HAM/LSC grafts were used to repair the cornea wounds by sutures (suture group, n=12) or by PTB technique (PTB group, n=12). The gross was observed including the corneal transparency, erythema, and new blood vessel formation after surgery. At 3 and 28 days, the inflammatory cytokine of interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α) were assayed by ELISA method; and the amount of new blood vessels were quantified by immunohistochemistry staining at 28 days. Results All animals survived to the end of the experiment. At 3 days, there was no obvious difference in the corneal transparency between 2 groups; at 28 days, the corneal transparency of PTB group was higher than that of suture group, and new blood vessels decreased. HE staining showed that mass inflammatory cells infiltrated between graft and cornea basal layer at 3 days, and no new blood vessel formed. inflammatory cells infiltration significantly decreased at 28 days in PTB group; the amount of new blood vessels was (2.0 ± 0.8)/ HP in PTB group and was (6.3 ± 1.3)/HP in suture group, showing significant difference (t=7.966, P=0.002). At 28 days, the concentrations of inflammatory cytokine of IL-1β, IL-6, and TNF-α in suture group were significantly higher than those in PTB group (P lt; 0.05); however, no significant differences were observed between 2 groups at 3 days (P gt; 0.05). Conclusion PTB technique can be used to fix HAM/LSC grafts, which can decrease inflammatory cell infiltration and new vessel formation, and improve the outcomes when compared with suture technique.
In order to understand how the biomechanical properties of rabbit cornea change over time after corneal ablation, 21 healthy adult rabbits were used in this study, with the left eye as experimental side and the right eye as the control side. Firstly, a lamellar knife was used to remove a portion of the anterior corneal surface tissue (30%~50% of the original corneal thickness) from the left eye of each rabbit, as an animal model simulating corneal refractive surgery. Secondly, postoperative experimental rabbits were kept for one, three, or six months until being euthanized. Strip specimens were produced using their corneas in vitro to perform a uniaxial tensile test with an average loading-unloading rate of approximately 0.16 mm/s. Finally, the visco-hyperelastic material constitutive model was used to fit the data. The results showed that there was a significant difference in the viscoelastic parameters of the corneas between the experimental and the control eyes at the first and third postoperative months. There was a difference in tangential modulus between the experimental and the control eyes at strain levels of 0.02 and 0.05 at the third postoperative month. There was no significant difference in biomechanical parameters between the experimental and the control eyes at the sixth postoperative month. These results indicate that compared with the control eyes, the biomechanical properties of the experimental eyes vary over postoperative time. At the third postoperative month, the ratio of corneal tangential modulus between the experimental and the control eyes significantly increased, and then decreased. This work lays a preliminary foundation for understanding the biomechanical properties of the cornea after corneal refractive surgery based on rapid testing data obtained clinically.
Objective To review research progress of corneal tissueengineering.Methods The recent articles on corneal tissue engineering focus on source and selection of corneal cells, the effects of growth factors on culture of corneal cells in vitro. The preparation and selection of three-dimensional biomaterial scaffolds and their b and weak points were discussed. Results The corneal tissue engineering cells come from normal human corneal cells. The embryo corneal cell was excellent. Several kinds of growth factors play important roles in culture, growth and proliferation of corneal cell, and incroporated into matrix.Growth factors including basic fibroblast growth factor, keratinocyte growth factor, transforming growth factor β1 and epidermal growth factor was favor to corneal cell. Collagen, chitosan and glycosaninoglycans were chosen as biomaterial scaffolds. Conclusion Human tissue engineering cornea can be reconstructed and transplanted. It has good tissue compatibility and can be used as human corneal equivalents.
ObjectiveTo investigate the feasibility of adipose-derived mesenchymal stem cells (ADMSCs) differentiating into corneal epithelium-like cells after transfection with Pax6 gene. MethodsThe adipose tissue from bilateral inguinal of healthy C57BL/6 mice (5-6 weeks old) was used to isolate and culture ADMSCs.The 3rd passage ADMSCs were subjected to treatments of non-transfection (group A),pcDNA3.1 empty vector transfection (group B),and recombinant plasmid of pcDNA3.1-Pax6 transfection (group C),respectively.At 48 hours after transfection,the cells in groups B and C were selected with G418.The cell morphology changes were observed under the inverted microscope.Pax6 protein and level of corneal epithelial cells specific molecular-cytokeratin 12 (CK-12) were measured by Western blot.Real-time fluorescence quantitative PCR was applied to measure the mRNA expression of CK-12. ResultsNo morphology change was observed in groups A and B.Two different cell clones were found in group C.No.1 selected clone showed a flagstone-like appearance that was similar to that of corneal epithelial cells;No.2 selected clone showed a net-like appearance,with 3-7 cell processes.The Western blot results showed the Pax6 protein expression in 2 clones of group C,but no expression in groups A and B; and CK-12 protein expression was only observed in No.1 selected clone of group C,and no expression in the others.The real-time fluorescence quantitative PCR results showed that the CK-12 mRNA expression level of No.1 selected clone of group C was 8.64±0.73,which was significantly higher than that of No.2 selected clone of group C (0.55±0.42),group B (1.36±0.40),and group A (1.00±0.00) (P<0.05),and there was no significant difference among groups A,B and No.2 selected clone of group C (P>0.05). ConclusionPax6 gene transfection could induce differentiation of ADMSCs into corneal epithelium-like cells which express CK-12 at both the mRNA and protein levels.This result provides a promising strategy of generating corneal epithelilcm-like cells for construction of tissue engineered cornea.
ObjectiveTo investigate the changes in the nerve fiber layer of the cornea in patients with demyelinating optic neuritis (DON) and its correlation with visual acuity. MethodsA cross-sectional study. From March 2021 to July 2022, 27 cases (39 eyes) of DON patients diagnosed in the Department of Neurology and Ophthalmology of Beijing Tongren Hospital Affiliated to Capital Medical University were enrolled in this study. According to the serological test results, the patients were divided into aquaporin 4 antibody associated optic neuritis (AQP4-ON group) and myelin oligodendrocyte glycoprotein antibody associated optic neuritis (MOG-ON group), with 15 cases (19 eyes) and 12 cases (20 eyes) respectively. According to previous history of glucocorticoid treatment, the patients were divided into glucocorticoid treated group and non-glucocorticoid treated group, with 17 cases (27 eyes) and 10 cases (12 eyes) respectively. Twenty healthy volunteers (20 eyes) with age- and gender-matched were selected as the control group. All eyes underwent best corrected visual acuity (BCVA) and in vivo confocal microscopy (IVCM) examinations. BCVA was performed using Snellen's standard logarithmic visual acuity chart, which was converted into logarithmic minimum angle resolution (logMAR) visual acuity during statistics. The corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve fiber branch length (CNBL), corneal nerve fiber branch density (CNBD) and the density of corneal dendritic cells (DC) were detected by IVCM examination. Parameter comparison between groups by t-test and Kruskal-Wallis rank sum test. The correlation between logMAR BCVA and pamameters of corneal nerve fibers were analyzed using Spearman analysis. ResultsThe CNFL, CNFD, and CNBL of the DON group and the control group were (10.67±2.55) mm/mm2, (57.78±12.35) root/mm2, (3.27±1.34) mm/mm2, and (13.74±3.05) mm/mm2, (70.95±13.14) root/mm2, and (4.22±1.03) mm/mm2, respectively; the difference in CNFL, CNFD, and CNBL between the two groups were statistically significant (t=4.089, 3.795, 2.773; P＜0.05). The CNFL, CNBL, and CNBD of the affected eyes in the MOG-ON group and AQP4-ON group were (12.02±2.13) mm/mm2, (3.80±1.19) mm/mm2, (47.97±8.86) fibers/mm2, and (9.25±2.19) mm/mm2, (2.72±1.19) mm/mm2, (39.43±13.86) fibers/mm2, respectively; the differences in CNFL, CNBL, and CNBD between the two groups were statistically significant (t=－4.002, －2.706, －2.306; P＜0.05). The corneal DC density of the patients in the hormone treated group and the non-hormone treated group was (24.43±8.32) and (41.22±9.86) cells/mm2, respectively. The difference in corneal DC density between the two subgroups was statistically significant (P＜0.001). Correlation analysis showed that there was a significant negative correlation between logMAR BCVA and CNBL and CNFL in patients with DON (r=－0.422, －0.456; P＜0.05). ConclusionsThere are different degrees of corneal nerve fiber damage in patients with different types of DON. There was a negative correlation between BCVA and the length of corneal nerve fibers.
Femtosecond laser small incision lenticule extraction (SMILE) with different residual stromal thicknesses (RST) is set to investigate its effect on corneal biomechanical properties of rabbits in vivo. In this study, 24 healthy adult Japanese rabbits were randomly divided into group A and B. The RST of group A was set 30% of the corneal central thickness (CCT), and the RST of group B was 50% of the CCT. The thickness of the corneal cap in both groups was set one third of CCT. Corneal visualization Scheimpflug technology (Corvis ST) and Pentacam three-dimensional anterior segment analyzer were used to determine corneal biomechanical and morphological parameters before surgery, and 1 week, 1 month and 3 months after surgery. Pearson correlation analysis was used to analyze factors affecting corneal biomechanical parameters after SMILE. The results showed that the corneal stiffness of group A was significantly higher than that of group B at 1 week and 1 month after surgery, and most biomechanical parameters returned to preoperative levels at 3 months postoperatively. The results of correlation analysis showed that postoperative CCT and RST were the main factors affecting corneal biomechanical parameters after SMILE. There was no significant difference in corneal posterior surface height (PE) between 3 months after surgery and before surgery in both two groups. It indicates that although the ability to resist deformation of cornea decreases in SMILE with thicker corneal cap and less RST, there is no tendency to keratoconus, which may be related to the preservation of more anterior stromal layer.
ObjectiveTo observe the changes in the biomechanical properties of the cornea of diabetic retinopathy (DR), and analyze its relationship with the degree of DR. MethodsA retrospective study. From September 2020 to February 2021, 83 patients with type 2 diabetes (T2DM) combined with DR treated in the Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, 83 eyes (DR group), 30 patients with T2DM without DR recruited from the outpatient clinic 30 eyes (NDR group) and 30 eyes of non-diabetes patients (NDM group) were included in the study. All left eyes were chose as the study eye. Among the 83 eyes in the DR group, 39 eyes were non-proliferative DR (NPDR) and 44 eyes were proliferative DR (PDR). Based on this, they were divided into NPDR group and PDR group. There was no statistically significant difference in age (t=1.10) and sex ratio (χ2=0.46) among patients in the DR group, NDR group, and NDM group (P>0.05); body mass index (t=3.74), glycosylated hemoglobin (t=35.02) and the length of the eye axis (t=5.51), the difference was statistically significant (P<0.05). The eye response analyzer (ORA) was used to measure the corneal hysteresis (CH), corneal resistance factor (CRF), Goldman related intraocular pressure (IOPg), and corneal compensatory intraocular pressure (IOPcc). The corneal topography was used to measure the central corneal thickness (CCT) of the examined eye. The differences of CCT, IOPcc, IOPg, CH, CRF among multiple groups were compared by one-way analysis of variance. Multiple linear regression was used to analyze the relationship between CH, CRF and related influencing factors in DR patients. ResultsThere were statistically significant differences in CCT, IOPcc, IOPg, CH, and CRF among the eyes of the DR group, NDR group, and NDM group (F=3.71, 5.60, 9.72, 9.02, 21.97; P<0.05). Pairwise comparisons were between groups, CH, CRF: the difference between the DR group and the NDM group and the NDR group was statistically significant (P<0.05); CCT: the difference between the DR group and the NDM group was statistically significant (P<0.05), and The difference in the NDR group was not statistically significant (P>0.05). CCT, CH, CRF: the difference between the NDR group and the NDM group was not statistically significant (P>0.05). The results of multiple linear regression analysis showed that CCT and IOPcc in DR patients were independent influencing factors of CH [CCT: β=0.01, 95% confidence interval (CI) 0.01-0.03, P=0.013; IOPcc: β=－0.15, 95%CI －0.25-－0.05, P=0.005]; Age, CCT, IOPcc were independent influencing factors of CRF [Age: β=－0.06, 95%CI －0.09-－0.03, P＜0.001; CCT: β=0.01, 95%CI 0.00-0.02, P=0.049; IOPcc: β=0.16, 95%CI 0.07-0.25, P=0.001]. The comparison of CCT, CH, CRF, adjusted CH, and adjusted CRF of the eyes in the NDR group, NPDR group, and PDR group were statistically significant (F=3.76, 5.36, 12.61, 6.59, 10.41; P<0.05). Pairwise comparison between groups, CH, CRF, adjusted CH, adjusted CRF: the difference between the NPDR group, the PDR group and the NDR group was statistically significant (P<0.05), and the difference between the PDR group and the NPDR group was not statistically significant (P>0.05); CCT: The difference between NPDR group and NDR group, PDR group and NPDR group was not statistically significant (P>0.05), and the difference between PDR group and NDR group was statistically significant (P<0.05). ConclusionThe CH and CRF of eyes with T2DM and DR are elevated; CCT and IOPcc are independent influencing factors of CH, and age, CCT and IOPcc are independent influencing factors of CRF.
Objective To investigate the influence of undercorrected orthokeratology on myopia control, and the correlation between target and central corneal epithelial damage. Methods A retrospective study was conducted on 22 undercorrected orthokeratology lens wearers (37 eyes) from January 2016 to February 2017, and 25 full corrected wearers (47 eyes) during the concurrent period were randomly selected as the control group. The changes of axial length before and after orthokeratology lens wearing and the within-6-month central corneal epithelial damage after orthokeratology lens wearing were analyzed. Results The average annual increase of axial length was (0.13±0.15) mm in the undercorrected group, and (0.14±0.16) mm in the full corrected group, the difference was not statistically significant (P>0.05). Multiple linear regression analysis showed that there was no correlation between the axial growth and the undercorrection of the target (P>0.05), but a negative correlation between the axial growth and the age (P<0.01). After using orthokeratology, the average annual growth of the axial length in children aged 7-10 years was (0.25±0.16) mm, and (0.10±0.14) mm in children aged 11-15 years, the difference was statistically significant (P<0.01). The incidence of central corneal epithelial punctate staining in the (–4.25)-(–5.00) D target group was 27.08%, and that in the (–3.00)-(–4.00) D target group was 16.67%, the difference was not statistically significant (P>0.05). Conclusions The effect of orthokeratology on myopia growth is not affected by the undercorrected target, not related to the undercorrection of target, but negatively correlated with the age. Undercorrected orthokeratology can still be used for myopia control in high myopia patients. No correlation is found between the target and central corneal staining.
Objective To investigate the clinical manifestations and gene mutation of a pedigree with retinal lattice degeneration and granular corneal dystrophy (GCD) type 2. Methods Ten members in 3 generations of a pedigree with retinal lattice degeneration and GCD2 were included in the study, including 6 patients (3 males and 3 females) and 4 healthy family members. All members underwent visual acuity, slit lamp microscope, three-mirror lens, fundus color photography, optical coherence tomography, and corneal endothelial cells counting. Genomic DNA was extracted from peripheral venous blood (2 ml) from all the subjects and their spouses, who had no related inherited diseases. The next generation sequencing method was used to detect the mutation sites of transforming growth factor β (TGFBI), and all results underwent Sanger verification. Results Among the 12 eyes of 6 patients, the visual acuity was FC/20 cm-1.0. In the superficial central corneal stroma, snowflake-like deposits were observed in three cases (6 eyes), and a small amount of granular deposits were observed in three cases (6 eyes). Corneal endothelial cell counts were normal. Retinal lattice degeneration were observed in 3 cases, 6 eyes (including 3 cases of rhegmatogenous retinal detachment in 4 eyes); retinal thinning without obvious lattice degeneration in 4 eyes of 2 patients. Nystagmus in 1 patient and fundus examination showed no significant abnormalities. DNA sequencing results showed that the proband and 4 patients had missense mutation of TGFBI gene in exon 4 c.371G＞ A, the mutation site corresponding to the amino acid change encoded by TGFBI gene No. 124 Amino acids, from arginine to histidine (p.R124H). Patients with this mutation have varying degrees of clinical phenotype. Conclusions The mutation of c.701G＞ A (p.R124H) in TGFBI gene is the causative gene of GCD in this pedigree. The patients with this mutation have different clinical phenotypes.
ObjectiveTo investigate whether the corneal shape recovered after discontinuation of long-term orthokeratology and whether orthokeratology increased the corneal astigmatism and ocular astigmatism.MethodsFrom December 2016 to April 2018, a retrospective study was conducted on 33 myopic patients who had undergone two times standard orthokeratology in the outpatient department of West China Hospital of Sichuan University, and had stopped wearing the first orthokeratology lens for one month before fitting the second orthokeratology lens. A total of 32 myopia frame glasses wearers were selected by simple random sampling as control. The changes of corneal flat meridian curvature (flat K), corneal steep meridian curvature (steep K), corneal astigmatism and ocular astigmatism before and after discontinuation of orthokeratology were analyzed.ResultsAmong the patients with baseline myopia diopter of −0.25～−2.75 D, the average annual change of corneal flat K was (−0.03±0.21) D in the frame glasses group and (−0.24±0.14) D in the orthokeratology group, the difference was statistically significant (t=5.555, P<0.001). Among the patients with baseline myopia diopter of −0.25～−2.75 D, the average annual change of corneal steep K was (0.20±0.42) D in the frame glasses group and (0.15±0.20) D in the orthokeratology group, the difference was not statistically significant (t=0.785, P=0.435). Among the patients with baseline myopia diopter of −3.00～−5.75 D, the average annual change of corneal steep K was (0.29±0.39) D in the frame glasses group and (−0.01±0.20) D in the orthokeratology group, the difference was statistically significant (t=2.758, P=0.014). The average changes of corneal astigmatism were analyzed according to the difference of eyes, gender, age and baseline corneal astigmatism, the difference was not statistically significant (P>0.05), respectively. For patients with baseline astigmatism absolute value less than or equal to 0.50 D, the astigmatism annual change of the frame glasses group was 0.00 (0.50) D, and that of orthokeratology group was −0.33 (0.48) D, the difference was statistically significant (Z=−2.301, P=0.021).ConclusionsThe flat K of the cornea becomes flatter and the steep K does not change after one month’s discontinuation of long-term orthokeratology. There was no difference in the increase of corneal astigmatism compared with those wearing frame glasses. When the baseline ocular astigmatism is less than or equal to 0.50 D, the increase of astigmatism may occur after discontinuation of orthokeratology.