Imaging and anatomical parameters of the lacrimal punctum and vertical canaliculus using optical coherence tomography.

Purpose: The anatomical parameters of normal lacrimal puncta and vertical canaliculus using optical coherence tomography (OCT) and the OCT imaging features of punctal lesions were analyzed to provide a basis for clinical diagnosis and treatment. Methods: From June to September 2019, 40 volunteers (80 eyes) from Tongji Hospital were enrolled. The external punctal diameter (ELP) was measured using slit-lamp microscopy and OCT. The internal lacrimal punctal diameter (ILP) at 100 µm, vertical canalicular length (VCL), and tear meniscus depth were measured by OCT with open eyes. Twenty-eight volunteers (56 eyes) underwent the same examinations with their eyes closed. The OCT imaging features of 26 patients (27 eyes) with lacrimal lesions were examined. Results: The ELP of the right and left healthy eyes under slit-lamp microscopy were 564.40 and 555.40 µm respectively. Under OCT, the ELP, ILP, and VCL of the right and left eyes were 628.20 um and 616.85 µm, 343.40 µm and 346.95 µm, 731.95 um and 709.20 µm respectively. The ELP was larger when measured by OCT than slit-lamp microscopy (p<0.05). Twenty-eight volunteers (56 eyes) had measurements taken under different conditions. The ELP, ILP, and VCL of the open and closed right eyes were 667.54 and 567.21 µm, 369.18 and 303.18 µm, 715.00 and 417.14 µm, respectively. The ELP, ILP, and VCL of the open and closed left eyes were 655.86 um and 551.68 µm, 369.25 um and 313.54 µm, 719.96 um and 433.89 µm respectively. The anatomical parameters of the open eyes were greater than those of the closed eyes (p<0.05). Thus, we identified the imaging features of lacrimal stenosis, punctal obstruction, punctal tear, lacrimal atresia, and lacrimal mass using OCT. Conclusions: OCT can be used to measure the anatomical parameters of lacrimal puncta and vertical canaliculus in vivo. In addition, OCT can detect punctal lesions in vivo and provide an objective basis for the clinical diagnosis and treatment of punctal lesions.

Microbiological contamination in donor corneas preserved for medium-term.

To evaluate the characteristics of microbiological contamination in donor corneas preserved for medium-term. A total of 82 donated corneas from June 1, 2014 to November 30, 2014 were retrospectively analyzed. The corneas were preserved in cornea chambers medium-term solution at 4-8 °C for keratoplasty. After removal of the central corneas for transplantation, the corneoscleral rims were put back into the medium for 1 month at room temperature (20-25 °C). The suspicious contaminated storage solutions indicated with transparency or color change were examined with bacteria and fungi cultivation for strain identification. The data collected included gender, age, procurement site and causes of death of donors, and follow-up of recipients. Statistical analysis was performed using Microsoft Excel and SPSS 24.0. Significance level was set at a P value < 0.05. The overall pathogen positive rate was 9.8% (n = 8), including 7 (87.5%) fungi and 1 (12.5%) bacteria. They were 2 (2.44%) Fusarium, 2 (2.44%) Chromomycosis, 1 (1.22%) Candida albicans, 1 (1.22%) Aspergillus versicolor, 1 (1.22%) Acremonium species, and 1 (1.22%) Enterococcus. 5 contaminated corneas were used for penetrating keratoplasty; although four out of five (80%) had not been given antifungal drugs during more than 6 months following-up period, none of the recipients was infected with a graft. Donor age (P = 0.839), gender (P = 0.062), procurement sites (P = 0.713) and cause of death (P = 0.711) had no statistically significant influence on the contamination rate. All donor corneas have a possibility of microbiological contamination. Strict tissue preservation protocol but not antifungal drugs following keratoplasty seems necessary to prevent graft infection.

A New Isolation Method of Human Lacrimal Canaliculus Epithelial Stem Cells by Maintaining Close Association with Their Niche Cells.

Purpose: To investigate whether lacrimal canaliculus epithelial stem cells (LCESC) could be isolated and expanded in vitro. Methods: The lacrimal canaliculus epithelium of 6 patients with limbal stem cell deficiency (LSCD) caused by alkali burn or Stevens Johnson Syndrome were examined by lacrimal endoscope. Cadaveric eyelids were fixed and prepared for cross section and stained with HE and antibodies against PCK, Vim, p63α, SCF and c-Kit. Canaliculus tissue was separated under an operating microscope using a lacrimal probe as an indicator and digested with collagenase A. The clusters of epithelial cells with closely associated stroma were further digested with Trypsin/EDTA to obtain single cells for culture on Matrigel-coated plastic plates in MESCM media. The expression of SCF, c-Kit and p63α was determined by immunostaining. The colony-forming efficiency on 3T3 feeder layers was also measured by calculating the percentage of the clone number divided by the total number cells seeded. Results: The epithelial layers of five out of six inferior lacrimal canaliculi and all the six superior lacrimal canaliculi were visually normal in appearance. Five to fifteen layers of the epithelium in the human lacrimal canaliculi were present with a small, tightly compacted basal layer of cells expressing PCK, p63α, SCF and c-Kit. LCESC were isolated by collagenase A and obtained clonal growth in MESCM. The colony-forming efficiency of LCESC holoclones on a 3T3 feeder layer was 3.2%, compared to 1.9% for those of limbal stem cells (LSC). Conclusions: Herein, we first report that LCESCs can be isolated and have stem cell characteristics, similar to those of LSCs. Such a discovery raises a promising substrate resource of stem cells for LSC reconstruction in LSCD patients.

Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation.

Corneal epithelial stem cells (SCs) are an ideal model for investigating how adult lineage-committed epithelial SCs are regulated by an anatomically defined and accessible niche, that is, limbal palisades of Vogt, located between the cornea and the conjunctiva. We have used collagenase digestion to isolate the entire limbal epithelial SCs and subjacent mesenchymal cells, and we have demonstrated that their close association is crucial for promoting epithelial clonal growth, implying that the latter serves as niche cells (NCs). After their close association was disrupted by trypsin/EDTA, single SCs and NCs could reunite to generate sphere growth in three-dimensional Matrigel in the embryonic SC medium, and that such sphere growth initiated by SC-NC reunion was mediated by SDF-1 uniquely expressed by limbal epithelial progenitor cells and its receptor CXCR4, but not CXCR7, strongly expressed by limbal stromal NCs. Inhibition of CXCR4 by AMD3100 or a blocking antibody to CXCR4 but not CXCR7 disrupted their reunion and yielded separate spheres with a reduced size, while resultant epithelial spheres exhibited more corneal differentiation and a notable loss of holoclones. For the first time, these results provide strong evidence supporting that limbal SC function depends on close physical association with their native NCs via SDF-1/CXCR4 signaling. This novel in vitro model of sphere growth with NCs can be used for investigating how limbal SC self-renewal and fate decision might be regulated in the limbal niche.

[Analysis of anatomy features for patients with narrow anterior chamber angle and primary iridociliary body cysts].

OBJECTIVE: To investigate the prevalence and anatomy features of iridociliary body cysts in patients with narrow anterior chamber angle. METHODS: Retrospective case series study. The prevalence and anatomy features of iridociliary body cysts in 223 patients (402 eyes) were analyzed retrospectively with ultrasound biomicroscopy (UBM). All of the patients were examined for susceptive narrow anterior chamber angle without complaint. The age of the patients, the site, diameter and number of cysts, the anterior chamber angle and the central anterior chamber depth were measured. RESULTS: Iridociliary body cysts were found in 19 patients (23 eyes) out of 223 patients (402 eyes), the prevalence is 5.7%. Fifteen patients were unilateral and four patients bilateral. Two cases originated from the ciliary process, eighteen cases from the iris root, and three from both the root and posterior surface of the iris. Twenty one cases were single cysts while two cases were multiple cysts. The diameter of the cysts ranged from 0.5 to 3.1 mm, averaged (0.71 ± 0.53) mm. The average age and the central anterior chamber depth of the eyes with iridociliary body cysts were (55.32 ± 10.74) years and (2.25 ± 0.39) mm, with no significant difference (t = 0.534, 0.783; P > 0.05) as compared to that of patients without cysts, which were (57.46 ± 10.52) years and (2.14 ± 0.34) mm. The anterior chamber angle in iridociliary body cysts group was 8.2° (21.0°, 0.0°), with no significant difference (Z = -0.062, P > 0.05) as compared to that of patients without cysts, which was 8.9° (21.4°, 0.0°). CONCLUSIONS: The prevalence rate of iridociliary body cysts in this study is 5.7%, central anterior chamber depth and anterior chamber angle in patients with cysts do not differ form patients without cysts.

Nicotinamide mononucleotide increases cell viability and restores tight junctions in high-glucose-treated human corneal epithelial cells via the SIRT1/Nrf2/HO-1 pathway.

BACKGROUND: Diabetes mellitus (DM)-related corneal epithelial dysfunction is a severe ocular disorder; however, the effects of nicotinamide mononucleotide (NMN) on high-glucose (HG)-treated human corneal epithelial cells (HCECs) remain unclear. METHODS: We conducted an in-vitro study to examine the effects of NMN treatment on HG-treated HCECs. Cell viability was measured using trypan blue stain, mitochondrial membrane potential was measured using JC-1 stain, and intracellular reactive oxygen species and apoptosis assays were conducted using flow cytometry. Transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) immunofluorescence for tight junction examinations were conducted. Immunoblot analyses were conducted to analyze the expression of silent information regulator-1 (SIRT1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) of the SIRT1/Nrf2/HO-1 pathway. RESULTS: NMN increased cell viability by reducing cell damage, reducing apoptosis, increasing cell migration, and restoring tight junctions in HG-treated HCECs. By analyzing the expressions of SIRT1, Nrf2, HO-1, NMN demonstrated protective effects via the SIRT1/Nrf2/HO-1 pathway. CONCLUSIONS: NMN increases cell viability by reversing cell damage, reducing apoptosis, increasing cell migration, and restoring tight junctions in HG-treated HCECs, and these effects may be mediated by the SIRT1/Nrf2/HO-1 pathway.

Redundant mechanisms for vascular growth factors in retinopathy of prematurity in vitro.

BACKGROUND: Current treatments for retinopathy of prematurity (ROP) targeting single vascular growth factors are ineffective in preventing neoangiogenesis. METHODS: We investigated the redundant/compensatory mechanisms between vascular growth factors in ROP. Cultured retinal vascular endothelial cells under CoCl2-induced hypoxia were transfected with recombinant adeno-associated virus type 2-vascular endothelial growth factor (VEGF) or pGIPZ-VEGF RNA interference to up- and downregulate VEGF expression, respectively. At 48 h after transfection, basic fibroblast growth factor (bFGF) and angiopoietin 1 (ANG1) gene expression as well as mitotic cycle changes were analyzed in the cells and correlated with the change in VEGF expression. RESULTS: Compared with the normal control group 1, at 30 min, 12 h and 24 h, the expressions of VEGF, bFGF and ANG1 in the hypoxia control group 2 were significantly higher. In the highly expressing VEGF group (group 3), the expressions of bFGF and ANG1 were downregulated, while in the low-expressing VEGF group 4, the expressions of bFGF and ANG1 were significantly upregulated. In the bevacizumab treatment group 5, the expressions of VEGF, bFGF and ANG1 were similar to those in group 2, and the difference was not significant. CONCLUSIONS: A compensatory mechanism (redundancy) exists between vascular growth factors in ROP. Such a phenomenon could partially explain why the inhibition of a single growth factor cannot effectively prevent the recurrence of neovascularization in ROP. A more effective strategy for treating ROP may be to inhibit VEGF and its redundant pathways.

[Analysis of multifocal electroretinogram first-order kernel P(1) wave in anisometropic amblyopia].

OBJECTIVE: To investigate the features of the multifocal electroretinogram (mfERG) first-order kernel P(1) wave in anisometropic amblyopic eyes. METHODS: mfERG of fifteen anisometropic amblyopic eyes and their normal control eyes were recorded with ROLAND RETIscan visual evoked response system, the mean amplitude density and eclipse period of the P(1) wave were analyzed and compared between the amblyopic and normal eyes with t-test. RESULTS: The P(1) wave amplitude density of mfERG first-order kernel in amblyopic eyes (164.7 ± 73.1) nV×deg(-2) was significantly attenuated in the central region of the visual field as compared with that of the control eyes (227.0 ± 61.3) nV×deg(-2) (t = 2.554, P = 0.016). The eclipse period of the amblyopic eyes (30.3 ± 4.3) ms was shorter than that of the control eyes (34.4 ± 3.2) ms (t = 2.92, P = 0.007). CONCLUSIONS: The significant change of the multifocal electroretinogram (mfERG) first-order kernel P(1) wave may reflect the abnormality of the retinal on-bipolar cells function and the visual information transmission, whether the P(1) wave could be used as an objective index of amblyopic eyes is a promising research topic.

[The influence of pressure on the cell survival and axonal growing in rat retinal ganglion cells in vitro].

OBJECTIVE: To evaluate the effect of pressures on the changes of purified cultivation of SD suckling rats' retinal ganglion cells (RGC) by a hyperbaric cell culture model and on the expression of neurofilament protein-H (NF-H) in vitro. METHODS: It was a control experimental study. RGC were purified from twenty SD neonatal rats (postnatal 1 - 3 days) using Thy1.1 antibody and cultured under the pressures of 0, 20, 40, 60, and 80 mm Hg (1 mm Hg = 0.133 kPa) respectively. The growth and survival times of RGC were observed, the numbers of processes of RGC were counted, and the processes measured under the different pressures after 48 h under phase-contrast microscope. The expression of NF-H and its distribution in primary purified RGC was investigated by an immunohistochemical technique and by semi-quantitative statistics analysis using microscopic image-analysis technique. The data was analyzed with SPSS 13.0 software. Difference among groups with One-way ANOVA and ambi-groups with SNK (q test), statistical significance was confirmed as P < 0.05. RESULTS: The purification rate of RGC reached 96.24% after cultured for 12 hours. Compared with the control group, the growth and survival times of RGC were similar (P = 0.595, 0.147) under the pressure of 20 mm Hg cultured for 48 hours, the expression of NF-H in RGC was not different (P = 0.227); but the growth and survival times of RGC were significantly different (P = 0.001, 0.000, 0.000 and 0.000, 0.000, 0.000) under the pressures of 40, 60, and 80 mm Hg, respectively. The expression of NF-H in RGC were significantly (P = 0.000, 0.000, 0.000) reduced under the pressures of 40, 60, and 80 mm Hg for 48 hours respectively. CONCLUSIONS: The growth and survival times of cultured RGC in vitro are affected by the pressure > 40 mm Hg leading to a decreased expression of NF-H indicating a pressure caused mechanism of RGC damage in glaucoma. Down-regulation of NF-H may influence the process growth of RGC.

[Expression of glucocorticoid receptors alpha and beta in mononuclear cells of peripheral blood in patients with glucocorticoid-induced ocular hypertension].

OBJECTIVE: To study the expression of glucocorticoid receptors (alpha and beta) in mononuclear cells of peripheral blood (PBMCs) in patients with glucocorticoid-induced ocular hypertension (GIOH) and the relationship between of the expression of GRalpha and GRbeta in PBMCs and its susceptibility to GIOH. METHODS: Case control study. Thirty-two patients with anaphylactic conjunctivitis were enrolled in this study. All of whom were received topical application of 0.1% dexamethasone. The patients with the elevation of intraocular pressure (IOP) over 8 mm Hg(1 mm Hg = 0.133 kPa) or higher within two weeks were defined as GIOH and the others were considered as control group. There were no statistical difference in sex, average age, and baseline IOP between two groups. Each group consisted of sixteen patients. GRalpha and GRbeta mRNA (relative value) in PBMCs were detected with semi-quantitative reverse transcriptional polymerase chain reaction (RT-PCR), GRalpha and GRbeta protein (positive ratio) in PBMCs examined with fluorescent labelling flow cytometry. RESULTS: The GRalpha mRNA/GAPDH ratio of GIOH group was significantly (t = 3.872, P < 0.05) higher than that of control group (1.152 +/- 0.057 vs 1.048 +/- 0.031). The GRbeta mRNA/GAPDH of GIOH group was lower than that of control group's (1.055 +/- 0.034 vs 1.063 +/- 0.035), but was not statistically different (t = 0.419, P > 0.05). GRalpha positive percentage was significantly (t = 8.513, P < 0.01) higher in PBMCs of GIOH group than that of the control group (93.10 +/- 7.35)% vs (46.00 +/- 13.11)%, whereas GRbeta positive percentage was significantly (t = 4.842 P < 0.01) lower in PBMCs of GIOH group than that in control group (12.50 +/- 4.18)% vs (23.83 +/- 3.92)%. CONCLUSIONS: The higher expression of GRalpha in PBMCs in patients may have an implication of susceptibility to GIOH and warrant further investigation.

P58(IPK) inhibition of endoplasmic reticulum stress in human retinal capillary endothelial cells in vitro.

PURPOSE: The goal of this research was to determine if P58(IPK), a member of the Hsp40 family that inhibits eukaryotic initiation factor 2alpha (eIF2alpha), inhibits endoplasmic reticulum (ER) stress and leads to downregulated expression of vascular endothelial growth factor (VEGF) and decreased apoptosis in human retinal capillary endothelial cells (HRCECs). METHODS: Recombinant vectors were constructed using P58 in adeno-associated virus type 2 (rAAV2-P58 (IPK)) and P58 RNA in the plasmid pGIPZ (pGIPZ-P58(IPK)). The four experimental groups were: (1) non-transfected/non ER stressed control; (2) non-transfected/ER stressed; (3) rAAV2-P58(IPK)-transfected/ER stressed; and (4) pGIPZ- P58(IPK) RNAi transfected/ER stressed. ER stress was induced by treating cells with tunicamycin. Expression of P58(IPK) was determined in transfected cells. Expressions of the following factors were assessed: vascular endothelial growth factor (VEGF), C/EBP homologous protein (CHOP), activating transcription factor 4 (ATF4), and glucose-regulated protein 78 (GRP78). Apoptosis levels were also determined. RESULTS: Significantly increased expression of P58(IPK) was detected in cells transfected with rAAV2-P58(IPK) (0.63+/-0.02) as compared to those transfected with pGIPZ-P58(IPK) RNAi (0.23+/-0.01). P58(IPK) expression was not different between the control transfected cells (rAAV2-GFP and pGIPZ-GFP). Following ER stress, expression levels of ATF-4, GRP78, CHOP, and VEGF in cells overexpressing P58(IPK) were not different from those in unstressed control cells. This inhibitory effect of P58(IPK) on the expression of ER stress-related factors was suppressed in cells transfected with pGIPZ-P58(IPK) RNAi. Apoptosis was significantly increased in cells transfected with pGIPZ-P58(IPK) RNAi but not with rAAV2-P58(IPK). CONCLUSIONS: The study demonstrates that P58(IPK) inhibits ER stress and plays an important role in maintaining balance and stability of the ER in HRCECs.

[Relationship between glucocorticoid receptors in the peripheral blood lymphocytes and trabecular meshwork and glucocorticoid induced glaucoma].

OBJECTIVE: To measure the number of glucocorticoid receptors (GR) in the trabecular meshwork and peripheral blood lymphocytes of rabbits, and to explore their relationship with steroid-induced glaucoma. METHODS: Steroid-induced glaucoma model was induced by subconjunctival injection of 0.5 mg dexamethasone in the right eyes every two days for a month. Before the injection, GR concentration was measured in peripheral blood lymphocytes and trabecular meshwork of rabbits using the radio-ligand binding assay. GR concentration was measured again 30 days after the injection. The data was analyzed with SPSS software. RESULTS: The GR concentration in the peripheral blood lymphocytes and the trabecular meshwork of rabbits was (3642 +/- 947) site/cell and (2437.85 +/- 733.93) dmp/mg, respectively. The expression level in lymphocytes was significantly correlated with that in the trabecular (r = 0.862, P < 0.01). After treated with dexamethasone, the intraocular pressure of rabbits increased significantly, and the GR concentration in peripheral blood lymphocytes decreased significantly. Elevating level of IOP correlated positively with the primary and decreasing level of GR (r = 0.78, 0.79, P < 0.01). CONCLUSIONS: GR concentration in the peripheral blood lymphocytes of rabbits can reflect directly the GR number in the trabecular meshwork. There may be a close relationship between the GR number in peripheral blood lymphocytes and steroid-induced glaucoma.

[Development of open cycling air pressure control system used for glaucoma research].

OBJECTIVE: To develop and set up a new culture system, which can apply pressure to cultured cells with open cycling air. The effects of this new system on the pH value, HCO(3)(-) concentration, O(2) pressure (pO(2)), CO2 pressure (pCO(2)) and the proliferation of retinal pigment epithelium (RPE) were tested to evaluate its efficiency in the study of glaucoma. METHODS: In the open cycling air pressure control culture system, the pressure inside the culture flasks was controlled by increase or decrease of the perfuse airflow. The influence of different culture systems (normal pressure culture system, open cycling air pressure control system and occlusive pressure control system) on the pH value, HCO(3)(-) concentration, pO(2), pCO(2) and proliferation of RPE were tested. The data were analyzed with SPSS software. RESULTS: The open cycling air pressure control culture system worked effectively, the pressure inside the culture flask can be controlled from 0 to 100 mm Hg. The difference of pH value, HCO(3)(-) concentration, pO(2), and pCO(2) of culture medium and the proliferation of RPE between normal pressure culture system and open cycling air pressure control system were not significant (P = 0.927, 0.887, 0.818, 0.770, 0.719, respectively). There was significant difference in these data between normal pressure culture system and occlusive pressure control system (P = 0.001, 0.000, 0.000, 0.000, 0.000, respectively). CONCLUSIONS: A new designed standard culture system applying pressure to cells with open cycling air was effective at pressure controlling and pH value, HCO(3)(-) concentration, pO(2) and pCO(2) controlling. This system may act as an ideal model in the experimental study of glaucoma.

[The inhibitory effects of angiostatin on retinal neovascularization induced by oxygen].

OBJECTIVE: To observe the inhibitory effects of angiostatin on microvascular endothelial cells of mouse retina and test the efficacy of native angiostatin in suppressing experimental retinal neovascularization induced by oxygen. METHODS: Angiostatin was purified with L-lysine Sepharose 4B from human plasma. The primary microvascular endothelial cells from rat retina were cultured. Microvascular endothelial cells growth inhibition assay was carried out with MTT method. Mouse models of hyperoxia-induced ischemic retinopathy were established. Angiostatin or normal saline (NS) were injected into the vitreous in 5 groups: normal, control and various doses. The nuclei of new vessel buds extending from the retina into the vitreous in different groups were counted and compared under the light microscope. RESULTS: Angiostatin could inhibit the growth of microvascular endothelial cells from rat retina in vitro. There were plenty of new vessel buds in the eyes of all mice in hyperoxic condition. The number of the nuclei of new vesselbuds in the murine eyes with injection of angiostatin. They were reduced by 42% (P < 0.01), 57% (P < 0.01) and 82% (P < 0.01) respectively. CONCLUSION: Angiostatin can powerfully inhibit growth of microvascular endothelial cells. The proliferation of retinal vessel may be suppressed by using angiostatin.

[The investigation of tranilast on the proliferation and migration of human Tenon's capsule fibroblasts].

OBJECTIVE: To investigate the effect of tranilast, N-(3,4-dimethoxycinnamoyl) anthramilic acid, on the proliferation and migration of human Tenon's capsule fibroblasts. METHODS: fibroblasts were cultured from Tenon's tissue of a glaucoma patient after trabeculectomy. The subcultured cells were incubated with DMEM medium containing different concentrations of tranilast for 72 h. The growth of fibroblasts was measured by methyl thiazlyl tetrazolium (MTT) assay and the cell count, and migration was evaluated by crutch method. The expression level of Protein kinase C (PKC) in fibroblasts was tested by immunohistochemical associated with image biological analysis (IBAS) methods. RESULTS: Treated with tranilast varying from 12.5 mg/L to 100.0 mg/L concentration, the proliferation of fibroblasts declined in a dose dependent manner. The migration of fibroblasts decreased from 40.20 +/- 5.83 to 22.50 +/- 4.21 and 9.80 +/- 2.14 cells/per field (P < 0.05) at 50.0 and 100.0 mg/L, respectively, and PKC expression was suppressed by tranilast from 0.2591 +/- 0.0038 to 0.2375 +/- 0.0106 and 0.1273 +/- 0.0573 Absorption value (P < 0.05) at 50.0 and 100.0 mg/L, respectively. CONCLUSION: Tranilast inhibited the proliferation as well as migration of fibroblasts in vitro, at least in part, by downregulation of PKC expression.

Amniotic membrane covering promotes healing of cornea epithelium and improves visual acuity after debridement for fungal keratitis.

AIM: To investigate the effect of amniotic membrane covering (AMC) on the healing of cornea epithelium and visual acuity for fungal keratitis after debridement. METHODS: Twenty fungal keratitis patients were divided into two groups randomly, the AMC group and the control group, ten patients each group. Both debridement of the infected cornea tissue and standard anti-fungus drugs treatments were given to every patients, monolayer amniotic membrane were sutured to the surface of the entire cornea and bulbar conjunctiva with 10-0 nylon suture for patients in the AMC group. The diameter of the ulcer was determined with slit lamp microscope and the depth of the infiltration was determined with anterior segment optical coherence tomography. Uncorrected visual acuity (UCVA) was tested before surgery and three month after healing of the epithelial layer. The healing time of the cornea epithelium, visual acuity (VA) was compared between the two groups using t-test. RESULTS: There was no statistical difference of the diameter of the ulcer, depth of the infiltration, height of the hypopyon and VA between the two groups before surgery (P>0.05). The average healing time of the AMC group was 6.89±2.98d, which was statistically shorter than that of the control group (10.23±2.78d) (P<0.05). The average UCVA of the AMC group was 0.138±0.083, which was statistically better than that of the control group (0.053±0.068) (P<0.05). CONCLUSION: AMC surgery could promote healing of cornea epithelium after debridement for fungal keratitis and lead to better VA outcome.

Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus.

AIM: To investigate whether the enhanced green fluorescent protein (EGFP) reporter gene could be transferred into human trabecular meshwork (HTM) cells by a HIV-based lentivirus both in vitro and ex vivo. METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection (MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1×10(8) transducing unit (TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining. RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo. Immunohistochemistry staining revealed that 88.19% EGFP-positive trabecular meshwork (TM) cells were observed in the human anterior segment. Nevertheless, the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group (P>0.05). CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.

Promotion of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium.

AIM: To investigate the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium (Ti) surface. METHODS: The chimeric peptide RKLPDAPRGDN (minTBP-1-PRGDN) was synthesized by connecting RKLPDA (minTBP-1) to the N-terminal of PRGDN, the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on Ti surface were tested using PRGDN and minTBP-1as controls. The keratocytes attached to the surface of Ti were either stained with FITC-labeled phalloidin and viewed with fluorescence microscope or quantified with alamar Blue method. The proliferation of keratocytes on Ti were quantified with 3-(4,5-dim- ethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide up-taking methods. The secretion of type I collagen were determined using an ELISA kit. RESULTS: The results showed that minTBP-1-PRGDN at a concentration of 100ng/mL was the most potent peptide to enhance the attachment of human keratocytes to the surface of Ti (1.40±0.03 folds, P=0.003), to promote the proliferation (1.26±0.05 folds, P=0.014) and the synthesis of type I collagen (1.530±0.128, P=0.008). MinTBP-1 at the same concentration could only promote the attachment (1.13±0.04 folds, P=0.020) and proliferation(1.15±0.06 folds, P=0.021), while PRGDN had no significant influence (P>0.05). CONCLUSION: Our data shows that the novel chimeric peptide minTBP-1-PRGDN could promote the attachment, proliferation and type I collagen synthesis of human keratocytes on the surface of Ti.

Isolation and expansion of human limbal stromal niche cells.

PURPOSE: Limbal stromal niche cells heterogeneously express embryonic stem cell (SC) markers. This study was conducted to isolate and expand them and to prove that their phenotype is critical for supporting SCs. METHODS: Human limbus was isolated by dispase or collagenase. Single cells were seeded on coated, 2D, or 3D Matrigel and were serially passaged in modified embryonic SC medium (MESCM), supplemented hormonal epithelial medium (SHEM), or Dulbecco's modified Eagle's medium plus 10% fetal bovine serum (DF) before they were seeded in 3D Matrigel. Sphere growth was achieved by mixing expanded single cells with dispase-isolated epithelial cells in 3D Matrigel. Expression of SC markers was analyzed by qRT-PCR, immunofluorescence staining, and Western blot; SC clonal growth was measured on 3T3 feeder layers. RESULTS: Collagenase, but not dispase, isolated subjacent mesenchymal cells, of which the expression of Oct4, Sox2, Nanog, Rex1, SSEA4, N-cadherin, and CD34 was promoted in MESCM more than SHEM or DF. Reunion of PCK+ and Vim+ cells generated spheres in 3D Matrigel, but spindle cells emerged on 2D or coated Matrigel. Serial passages on coated Matrigel resulted in rapid expansion of spindle cells, of which the expression of ESC markers had declined but could be regained after reseeding in 3D Matrigel in MESCM but not in SHEM or DF. Resultant epithelial spheres mixed with spindle cells expanded in MESCM expressed more p63α, less CK12, and more holoclones than those mixed with spindle cells expanded in DF. CONCLUSIONS: Limbal stromal niche cells expressing SC markers can be isolated and expanded to prevent differentiation and maintain clonal growth of limbal epithelial progenitors.

Expression of endoplasmic reticulum stress-related factors in the retinas of diabetic rats.

Recent reports show that ER stress plays an important role in diabetic retinopathy (DR), but ER stress is a complicated process involving a network of signaling pathways and hundreds of factors, What factors involved in DR are not yet understood. We selected 89 ER stress factors from more than 200, A rat diabetes model was established by intraperitoneal injection of streptozotocin (STZ). The expression of 89 ER stress-related factors was found in the retinas of diabetic rats, at both 1- and 3-months after development of diabetes, by quantitative real-time polymerase chain reaction arrays. There were significant changes in expression levels of 13 and 12 ER stress-related factors in the diabetic rat retinas in the first and third month after the development of diabetes, Based on the array results, homocysteine- inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1(HERP), and synoviolin(HRD1) were studied further by immunofluorescence and Western blot. Immunofluorescence and Western blot analyses showed that the expression of HERP was reduced in the retinas of diabetic rats in first and third month. The expression of Hrd1 did not change significantly in the retinas of diabetic rats in the first month but was reduced in the third month.