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.
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.
In order to rescue the serious cornea-sclera defect, normal piece of a sclera from the damaged eye was used to serve as a donor material to repair the cornea-sclera defect. Eighteen cases were treated by transplantation of sclera graft with the operation to decompress the intra-ocular tension and to perform an artificial pupil from 1979 to 1994. Because the ocular-store seriously lacks in mostly internal locality, in the 18 cases, laceration of avulsive in 5 cases, severe corneal fistula complicated to other corneal diseases in 8 cases, locolized staphyloma of aornea in 2, laceration of sclera fistula in 1. The resulte were: The repairs were all healed up in an average of 20 days. The tension returned to normal and the vision was improved. The scleral graft was fused with the cornea, and the white area form the graft tended to reduce in size gradually with increase in transparency. There was no sign of iritis and rejection reaction. Follow-up of 3 to 36 months showed that the grafting operation was simple and an easy method, at the same time, once gained effect if would prevent or cure blindness.
Objective To observe the efficacy of photodynamic therapy for vitelliform macular dystrophy(VMD) with choroidal neovascularization(CNV). Methods The clinical data of 7 patients (7 eyes) of VMD with CNV who had undergone photodynamic therapy (PDT) were retrospectively analyzed. The patients were 4 males and 3 females, aged from 20 to 54 years. The patients received the examinations of best corrected visual acuity (BCVA), slitlamp microscopy, fundus photography, fluorescein angiography (FFA), indocyanine green angiography (ICGA), spectral domain OCT(SD-OCT), electrooculogram（EOG）and electroretinogram (ERG)before and after PDT. The BCVA ranged from finger counting to 0.6. Retinal edema and the subretinal fluid were observed. The mean thickness of central retina was (506.00plusmn;30.71) mu;m. PDT was performed according to the standard treatment. The follow-up period ranged from 2 to 11 months with the mean of 6.3 months. The changes of BCVA, CNV and side effects were observed after treatment. Results BCVA improved in all patients ranging from 0.12 to 1.0. The regression of the CNV and resolution of the subretinal fluid were observed by FFA, ICGA and SD-OCT after PDT. The mean thickness of central retina was reduced to (401.00plusmn;52.22) mu;m. There was no PDTassociated ocular or systemic side effect. Conclusions PDT is an effective and safe treatment for VMD with CNV. It may improve or stabilize the visual acuity.
Objective To evaluate glaucoma and anterior chamber angle changes after intravitreal silicone oil injection(SOI). Methods The intraocular pressure(IOP) and anterior chamber angles of 34 e yes(33 patients)undergone pars plana vitrectomy and SOI were observed. Results Glaucoma occurred in 9 eyes(26%),in which silicone oil glaucoma(SOG) accounted for 77%(7/9).The changes of the superior part of anterior chamber angle were peripheral anterior synechiae in 11 eye(32%) and SO emulsification droplets in 22 eyes(64%) in 1～4.5 months after surgeries.Glaucoma occurred in 6 eyes of 7 eyes undergone peripheral iridectomy at the inferio r part of the iris after the closure of iridectomy holes (plt;0.05). Conclusion High incidence of glaucoma was found in this series of patients after intravitreal silicone oil injection,and the main causes of SOG were closure of the inferior iridecomy hole and silicone emulsification. (Chin J Ocul Fundus Dis, 2001,17:105-107)
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.
Objective To observe the effects on rabbit corneas and retinas after single intravitreal injection of voriconazole at different doses. Methods According to the randomization table, 25 healthy rabbits were randomly divided into control group, and voriconazole 50, 100, 200, and 400 μg groups. Therefore, there were 5 rabbits in each group. The eyes of control group received intravitreal injection of 0.1 ml balanced saline solution, and those treatment groups received 0.1 ml voriconazole injection of corresponding dose. Before the injection and 1, 7, and 14 days after the injection, endothelial cell counts and corneal thicknesses were measured; full-field electroretinogram were performed and b-wave amplitudes in maximal combined reaction (Max-R) were recorded. On 14 days after the injection, histologic structures were observed by light microscope and transmission electron microscope. Results There was no significant difference in endothelial cell counts (F=0.320, 0.291, 0.467, 0.649) and corneal thicknesses (F=0.214, 0.284, 0.360, 0.225) with those of control group at any time points (P > 0.05). Before and 1 day after the injection, b-wave amplitudes of each voriconazole group had no significant difference compared with those of control group (F=0.220, 0.106; P > 0.05). On 7 days after the injection, b-wave amplitudes decreased significantly at doses of 200 μg and 400 μg (P < 0.05). On 14 days after the injection, there was no significant difference between the the amplitude of 200 μg group and that of control group (P > 0.05). However, the amplitude of the 400 μg group decreased continuously and there was still significant difference (P < 0.05). Light microscopy did not reveal any corneal abnormality in both control group and voriconazole groups. The retinas were normal except that of the 400 μg group, which hadathinner and degenerated inner nuclear layer and disordered photoreceptor layer. Under transmission electron microscope, there were no ultrastructure damages of corneas in both control group and voriconazole groups, either. The rabbit retinas of the 50 μg and 200 μg group have normal inner nuclear layer and photoreceptor layer, but degrees of changes in both layers were observed in the eyes of 200 μg and 400 μg group. Conclusions There is no obvious effects on rabbit corneas and retinas after single intravitreal injection of voriconazole at he dose less than or equal 100 μg. There are no obvious effects on rabbit corneas at the dose of 200 μg and 400 μg, while there are damages to the retinas in both functions and histological structures.
Objective To observe the corneal nerve fibres damage in different stage of diabetic retinopathy (DR) with type 2 diabetes. Methods A cross-sectional study. One hundred and twenty eyes of 120 patients with type 2 diabetes served as diabetes group. According to International Clinical Diabetic Retinopathy Disease Severity Scales (2002), diabetes patients were classified into 4 subgroups: patients without diabetic retinopathy (NDR), patients with mild or moderate non-proliferative diabetic retinopathy (mNPDR), patients with severe non-proliferative diabetic retinopathy (sNPDR) and patients with proliferative diabetic retinopathy (PDR), each subgroup has 30 eyes of 30 patients. Another 30 eyes of 30 healthy participants served as control group. All eyes were scanned with HRT3 in vivo corneal confocal microscopy. Images of sub-basal nerve plexus were quantified including nerve fiber length (NFL), nerve fiber density (NFD), nerve fiber branch density (NFB), and nerve tortuosity (NT). The correlations of corneal nerve fiber with age, duration of diabetes and glycated hemoglobin (HbA1c) were analyzed using Spearman correlation analysis. Results NFL, NFD and NFB were found to be significantly lower in diabetic patients (F=147.315, 142.586, 65.898;P=0.000, 0.000, 0.000), NT was significantly greater in diabetic patients (F=39.431,P=0.000), when compared to control group. In diabetic patients, NFL, NFD and NFB were gradually reduced with DR severity, NT was gradually increased with DR severity. While the difference of NFL, NFD, NFB, NT was not statistically significant between sNPDR and PDR subgroups (P＞0.05), but was statistically significant between other subgroups (P＜0.05). Spearman correlation analysis results showed that age (r=－0.071, －0.080, 0.001, 0.100;P=0.391, 0.328, 0.991, 0.224) and HbA1c (r=－0.109, －0.115, －0.126, 0.025;P=0.238, 0.211, 0.169, 0.781) had no correlation with NFL, NFD, NFB, NT. Duration of diabetes was negatively correlated with the NFL, NFD (r=－0.212, －0.264;P= 0.020, 0.004), positive correlated with NT (r=0.261,P=0.004), and had no correlation with NFB (r=－0.119,P=0.194). Conclusions Corneal nerve fiber loss and nerve tortuosity increased were found in patients with type 2 diabetes, and even without diabetic retinopathy. The progress of corneal neuropathy was correlated with the severity of DR, but it was not change significantly between sNPDR and PDR.
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.
Objection To compare the effects of three mainstream surgical methods on the ocular surface and investigate the optimal corneal myopia correction surgery. Methods We selected 118 patients (236 eyes) undergoing small incision lamellar extraction (SMILE) (40 patients, 80 eyes), femtosecond-laser in situ keratomileusis (FS-LASIK) (36 patients, 72 eyes) or sub-Bowman keratomileusis (SBK) (42 patients, 84 eyes) surgery for corneal myopia correction from March 2015 to January 2016. Before surgery, one week and one month after surgery, tear film rupture time, tear river height and eye red index were measured using ocular surface analyzer. The Schimer Ⅰ test was used to detect tear secretion. Corneal sensory gauge was used to measure corneal sensation. Analysis of variance was used to analyze the measurements of the three surgical methods at various time points. Results The tear film rupture time of the three groups decreased after one week (P<0.05), but the time in the SMILE group [(9.643±4.751) seconds] was longer than those in the other two groups [FS-LASIK (8.172±4.300) seconds, SBK (7.612±3.691) seconds,P<0.05]. The time in the SMILE group at one month after surgery was not different from that before surgery (P>0.05). At one month after surgery, the rupture times of the other two groups were still shortened (P<0.05). The tear river heights in the three groups were decreased at one week and one month after surgery (P<0.05), except FS-LASIK group (P>0.05). In the SBK group, at one week after surgery, the decrease of the height was the most obvious (P<0.05). There was no difference in eye red index among the three groups at the time of observation (P>0.05). Corneal sensation decreased in the three groups at one week and one month after surgery. In the SBK group, that was the most obvious decline (P<0.05). In the three groups, the tear secretion did not change at each observation time point (P>0.05). Conclusion In the three surgical methods, SMILE has the minimal impact on the ocular surface, followed by FS-LASIK, and SBK has the greatest impact.