Clinical outcomes of different regimens of intravitreal Conbercept for the treatment of choroidal neovascularization secondary to pathological myopia.

PURPOSE: To assess the clinical outcomes of two intravitreal injection regimens of Conbercept used to treat choroidal neovascularization secondary to pathological myopia (PM-CNV). METHODS: A total of 72 eyes of 72 patients were treated: 39 eyes received a single injection followed by treatment pro re nata (1 + PRN); 33 eyes first received 3 consecutive monthly injections (3 + PRN) then followed by PRN. After initial injection, patients were followed up for 12 months. RESULTS: The mean age of 72 patients was 45.3 ± 5.1 years, with the mean diopter of -10.62 ± 3.24D. The best corrected visual acuity (BCVA) was 0.86 ± 0.23 LogMAR with 1 + PRN and 0.90 ± 0.19 LogMAR with 3 + PRN at baseline (P = 0.422), 0.36 ± 0.07 and 0.33 ± 0.05 LogMAR at month 3 (P = 0.026); and 0.33 ± 0.03 and 0.32 ± 0.02 LogMAR at month 12 (P = 0.096). The central retinal thickness (CRT) was 333.5 ± 22.7 µm with 1 + PRN and 341.2 ± 20.9 µm with 3 + PRN at baseline (P = 0.139), 281.53 ± 10.28 and 273.15 ± 13.24 µm at month 3 (P = 0.004); 266.83 ± 8.14 and 264.91 ± 9.27 µm at month 12 (P = 0.350). The number of injections in the 1 + PRN group was significantly lower than that observed in the 3 + PRN group (2.15 ± 1.06 versus 3.36 ± 0.74; P < 0.001). During the follow-up, no serious ocular complications and adverse reactions related to Conbercept and injections occurred. CONCLUSIONS: Both injection regimens resulted in similar visual outcomes in PM-CNV patients. The 1 + PRN regimen had fewer injections and might be more suitable in this patient population.

A modified high-yield method for primary culture of rat retinal microglial cells.

Microglial cells are the main immune cells of the retina. The primary culture of the retinal microglia is critically important in investigating the cells' properties and behaviors in neurodegenerative and inflammatory retinal disease. Here, we described a modified protocol of a microglial cell culture from the neonatal rat retina. In our culture protocol, the retina was isolated from the neonatal rat eye from postnatal day 1 to day 3 and trypsinized into a single-cell suspension. The cells were seeded into a T75 flask, which was pre-coated with poly-D-lysine (PDL) and cultured with dulbecco's modified eagle medium-F12 (DMEM/F12) that contained 10% fetal bovine serum (FBS) with different concentrations. Small bright rounded cells were observed on the top of mixed glial cells on the seventh day, and attained the maximum cell number on the 14th day. Then, the isolation was performed by a shaking method and isolated cells were identified with microglia markers ionized calcium-binding adaptor molecule 1 (IBA1), transmembrane protein 119 (TMEM119), cluster of differentiation 11b (CD11b), as well as astrocyte marker glial fibrillary acidic protein (GFAP) by immunofluorescence staining. Additionally, the initial plating ratio of the mixed glial cell, culture period of isolation, procedures of the isolation, as well as the purification procedure, were optimized for our primary microglial cell culture. The morphological changes and phagocytic function were performed after lipopolysaccharide (LPS) stimulation. Moreover, the release of pro-inflammatory cytokines at different time points of LPS activation were measured. In the present study, we found that the concentration of one retina/T75 flask could harvest the largest number of microglial cells. Besides, we continuously cultured the mixed glial cells as long as one month and isolated the mixed glial cells as much as three times. In our study, we used an isolation-shaking rate of 200 rpm for 2h, which guaranteed the steady rate and resulted in high purification of the primary retinal-microglial cells, with no need of an additional purification procedure. In conclusion, we provided a high-producing protocol for the primary culture of purified rat retinal-microglial cells.

Combined central retinal vascular occlusion as the presenting feature in ß-thalassemia with iron deficiency anemia.

PURPOSE: To report a case of ß-thalassemia trait (ß-TT) with iron deficiency anemia (IDA) presenting as a combined central retinal vein and artery occlusion (CCRVAO). METHODS: Case report. A 22-year-old female presented with sudden-onset blurry vision in the left eye of 3-days duration. RESULTS: Best corrected visual acuity was 20/20 and 20/1000 in right and left eyes, respectively. Fundus examination of left eye revealed optic disc edema, macular whitening with a cherry-red spot, markedly dilated and tortuous retinal veins, and hemorrhages both around the disc and extending into the macula and the periphery. Fundus fluorescein angiography (FFA) showed delayed filling of retinal vasculature, dilated and tortuous retinal veins, blocked fluorescence around and beyond the optic disc. OCT scan at presentation showed hyperreflective inner retinal layers with neurosensory detachment. OCTA showed that the vessel densities of superficial and deep capillary plexus were remarkably reduced.A diagnosis of ß-TT combined with IDA was made after hematologic work-up. The patient was treated with a course of oral iron supplements, vasodilator (Compound Xueshuantong), inhalation of a mixture of 5% carbon dioxide and 95% oxygen, and a nutritional agent (compound anisoine). By six months later, her visual acuity improved to 20/60 in the left eye with complete resolution of all clinical signs. CONCLUSION: CCRVAO is a rare emergency leading to acute vision loss and can manifest in patients with ß-TT with IDA. Prompt diagnosis and early management is important to treat underlying systemic disorders and to prevent occurrence of a similar episode in fellow eye.

Human Amniotic Epithelial Cells Promote the Proliferation of Human Corneal Endothelial Cells by Regulating Telomerase Activity via the Wnt/ß-catenin Pathway.

PURPOSE: Human amniotic epithelial cells (HAECs) have regenerative properties and low immunogenicity, which have enabled their use without immune rejection in regenerative medicine applications, such as wound repair, corneal surgery and burn repair. The aim of this study was to explore the potential role of HAECs in the proliferation of human corneal endothelial cells (HCEnCs) and the possible mechanism of regulation. METHODS: HAECs and HCEnCs were isolated from donated tissue samples and were cultured; the collected HAEC culture medium (HAEC-Me) was added to the human corneal endothelium medium (CEM) to establish the HAEC-CM system. HCEnCs were cultured in CEM, 20%HAEC-Me, 20% HAEC-CM, 20% HAEC-CM supplemented with a GSK-3ß inhibitor TWS119 or CEM supplemented with TWS119. Then, cell proliferation, apoptosis, cell cycle progression, telomerase activity, and Wnt/ß-catenin pathway-related protein levels were assessed. RESULTS: We found that the HCEnCs cultured in the 20% HAEC-CM had increased proliferative capacity, telomerase activity and ß-catenin and Tcf4 expression levels, and they had a decrease in the rate of apoptosis and α-SMA expression when they were compared with the HCEnCs cultured in the 20% HAEC-Me. After GSK-3ß was inhibited by TWS119, HCEnCs cultured in CEM or 20% HAEC-CM had an increased proliferative capacity, telomerase activity, ß-catenin/Tcf4 expression and a decreased α-SMA expression, and they had a decreased apoptotic rate. CONCLUSIONS: These data indicate that the human amniotic epithelial cells microenvironment can promote the proliferation of human corneal endothelial cells, which may be related to regulating telomerase activity and epithelial-to-mesenchymal transition (EMT) via the Wnt/ß-catenin pathway.

Transient effect of suction on the retinal neurovasculature in myopic patients after small-incision lenticule extraction.

PURPOSE: To characterize retinal neurovasculature changes after small-incision lenticule extraction (SMILE) in myopic patients. SETTING: Ophthalmic Center, the Second Affiliated Hospital of Guangzhou Medical University, China. DESIGN: Prospective interventional study. METHODS: The corrected distance visual acuity/uncorrected distance visual acuity, corrected intraocular pressure (CIOP), and corneal tomography were evaluated at baseline (PRE), postoperative day (POD) 1, and POD 7. Ganglion cell-inner plexiform layer (GCIPL) and peripapillary retinal nerve fiber layer (pRNFL) thicknesses were measured. The vessel area densities (VADs, %), vessel skeleton densities (VSDs, %), vessel diameter index (VDI), and fractal dimensions (Dbox) of the superficial vascular plexus (SVP) and deep vascular plexus (DVP) were measured in a circular area (ϕ 2.5 mm) centered on the fovea. RESULTS: A total of 38 myopic patients were recruited. The GCIPL thickness was increased after SMILE at POD 1 and POD 7 (P < .01) but no significant changes in the pRNFL thickness. The VAD, VSD, and Dbox of the SVP were decreased at POD 1 (P < .01), but not at POD 7. The VDI in small vessels of the SVP and DVP was decreased at POD 1 (P < .05) and increased at POD 7 (P < .05). Changes in CIOP were positively correlated with changes in the GCIPL thickness. Changes in CIOP were negatively correlated with changes in the VAD of small vessels and the Dbox of total vessels in the DVP. Changes in CIOP were negatively correlated with the VSD and VDI of small vessels in the DVP and changes in the VDI of big vessels in the SVP. CONCLUSIONS: The transient fluctuations in the retinal neurovasculature after SMILE may represent a characteristic homeostasis pattern in patients after refractive surgery.

Pterygium epithelium abnormal differentiation related to activation of extracellular signal-regulated kinase signaling pathway in vitro.

AIM: To investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase (ERK) signaling pathway in vitro. METHODS: The expression levels of phosphorylated ERK (P-ERK), keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays, the differences in clone morphology and in K10, K19, P-ERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency, differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis. RESULTS: The expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium. Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19. The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with 3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression. CONCLUSION: We suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation.

Role of Retinal Nerve Fiber Layer Thickness and Optic Disk Measurement by OCT on Early Diagnosis of Glaucoma.

PURPOSE: Glaucoma is an eye disease that can lead to irreversible optic nerve damage and cause blindness. Optical coherence tomography (OCT) allows an early diagnosis of glaucoma by the measurements of the retinal nerve fiber and optic disc parameters. A retrospective study was designed to analyze the effects of the measurement of the retinal nerve fiber layer (RNFL) thickness and the optic disc tomography by spectral-domain OCT on the early diagnosis of suspected glaucoma and primary open angle glaucoma (POAG). METHODS: This was a clinical case-control study. The RNFL thickness around the optic disc and optic disk tomographic parameters of the control (n = 51, 98 eyes), suspected glaucoma (n = 81, 146 eyes), and POAG groups (n = 55, 106 eyes) were measured by OCT. The parameters included superior, inferior, nasal and temporal mean RNFL thickness, disc area (DA), cup area (CA), rim area (RA), disc volume (DV), cup volume (CV), rim volume (RV), cup/disc area ratio (CA/DA), rim/disc area ratio (RA/DA), cup/disc volume ratio (CV/DV) and rim/disc volume ratio (RV/DV). RESULTS: Superior, nasal, and mean RNFL parameters, DA, CA,RA, DV, CV, CA/DA, RA/DA, CV/DV and RV/DV significantly differed among three groups by single-factorial ANOVA. Inferior and temporal RNFL thickness significantly differed between the control and POAG groups. No significant difference was observed in RV among three groups. In the POAG group, the maximum area under the ROC curve (AROC) of mean RNFL thickness was 0.845. The maximum AROC of optic disk parameters was RA/DA (0.998), followed by CA/DA (0.997). The AROC of CA, RA, CV, and DV were all > 0.900. CONCLUSION: OCT may serve as a useful diagnostic modality in distinguishing suspected glaucoma from POAG.

Inhibition of α-smooth muscle actin expression and migration of pterygium fibroblasts by coculture with amniotic mesenchymal stem cells.

PURPOSE: Epithelial-mesenchymal transition (EMT) of normal conjunctival tissues is a major reason of pterygium generation; α-smooth muscle actin (α-SMA) is a maker in EMT. In this study, we investigated if human amniotic mesenchymal stem cells (hAMSCs) can inhibit α-SMA expression and migration of human pterygium fibroblasts (hPFs) in vitro. MATERIALS AND METHODS: Coculture of hAMSCs and hPFs was completed by using a Transwell coculture system. The α-SMA expression of hPFs was detected by immunocytochemistry and western blot analysis. The migration ability of hPFs was measured by means of a migration assay. Immunoreactivity for α-SMA was detected in all pterygium fibroblasts examined. RESULTS: The expression of α-SMA in cocultured hPFs was significantly lower than in hPFs. Similar result was demonstrated in western blot analysis. In addition, migration assay showed that hAMSCs reduce the migration of hPFs. CONCLUSIONS: These results suggested that hAMSCs have the potential to inhibit the generation and invasiveness of pterygium in vitro.

Relationship between Foxp3-3279 (rs376158) polymorphism and dust mite allergic conjunctivitis.

PURPOSE: To investigate the genotyping of Foxp3-3279 (A/Crs376158) genes in patients with dust mite-induced allergic conjunctivitis from Guangdong province and to explore the association between these genes and the susceptibility to dust mite allergic conjunctivitis. METHODS: In total, 80 patients with dust mite allergic conjunctivitis and 103 healthy Han Chinese were enrolled in the study and received genotyping of Foxp3-3279 (A/C,rs376158) by PCR-SSP technique. RESULTS: Genotype frequency of Foxp3-3279 AA, CA, and CC in patients with dust mite allergic conjunctivitis were 1.25%, 25.00% and 73.75%,respectively. Gene frequency of C and A in patients with dust mite allergic conjunctivitis were 86.25% and 13.75% with no significant difference from healthy counterparts (both P > 0.05). CONCLUSION: Foxp3-3279 polymorphisms did not significantly differ between patients with dust mite allergic conjunctivitis and healthy controls, implying that this genetic locus is probably not an independent risk factor of the underlying pathogenesis of dust mite allergic conjunctivitis.

Toric intraocular lens vs. peripheral corneal relaxing inci- sions to correct astigmatism in eyes undergoing cataract surgery.

PURPOSE: To compare toric intraocular lens implantation (Toric-IOL) with peripheral corneal relaxing incisions (PCRIs) for astigmatism correction in patients undergoing cataract surgery. METHODS: 54 patients (54 eyes) with more than 0.75 diopter (D) of preexisting corneal astigmatism were classified as group A (0.75-1.50D) or group B (1.75-2.50D). The patients were randomized to undergo Toric-IOL or PCRIs in the steep axis with spherical IOL implantation. LogMAR uncorrected visual acuity (LogMAR UCVA), LogMAR best corrected visual acuity (LogMAR BCVA), error of vector (|EV|), surgery induced refraction correction (|SIRC|), and correction rates (CR) were measured 1 month and 6 months postoperatively. RESULTS: At 6 months postoperatively, all 54 eyes had LogMAR BCVA ≤ 0.2. Patients who underwent PCRIs and Toric-IOL with LogMAR BCVA ≤ 0.1 showed no significant differences in group A (P = 1.00) or in group B (P = 0.59). Group A showed no significant differences in LogMAR UCVA (P = 0.70), |EV| (P = 0.13), |SIRC| (P = 0.71), and CR (P = 0.56) in patients underwent PCRIs and Toric-IOL. However, group B showed significant differences in LogMAR UCVA (P < 0.01), |EV| (P < 0.01)), |SIRC| (P < 0.01), and CR (P < 0.01). The LogMAR UCVA and |EV| between 1 and 6 months showed no significant differences in patients in group A. However, in group B, they are significant differences. CONCLUSION: The efficacy and stability of Toric-IOL and PCRIs were equal in low astigmatic patients. Toric-IOL achieved an enhanced effect over PCRIs in higher astigmatic patients. PCRIs had the more refractive regression than Toric-IOL in 6 months.

Effect of Avastin on the migration and invasion of pterygium fibroblasts.

PURPOSE: To evaluate the effect of Avastin on human pterygium fibroblast migration and invasiveness. METHODS: VEGF secretion was compared between human pterygium fibroblasts and conjunctival fibroblasts by measuring VEGF-A by ELISA. The influence of Avastin on HPF migration and invasiveness was observed by wound scratch and Transwell migration assays. The expression of p-ERK1/2 and p-FAK was analyzed by western blotting. RESULTS: (1)VEGF was secreted in higher amounts by human pterygium fibroblasts than by conjunctival fibroblasts. (2) Avastin treatment decreased HPF migration and invasion. (3) Avastin significantly decreased the expression of p-ERK1/2 and p-FAK in human pterygium fibroblasts. CONCLUSION: Avastin can inhibit migration and invasion of HPFs by decreasing the expression of p-ERK1/2 and p-FAK.

Human amniotic epithelial cell niche enhances the functional properties of human corneal endothelial cells via inhibiting P53-survivin-mitochondria axis.

The particular microenvironment or niche plays an important role in determining the fate of stem cells and adult cells. The objective of this study was to explore the potential role of the niche of human amniotic epithelial cells in enhancing the functional properties of human corneal endothelial cells (HCECs). The HCECs were cultured in different media, including corneal endothelium medium (CEM), 20% human amniotic epithelial cell culture medium (20% HAEC-Me), and 20% human amniotic epithelial cell-conditioned medium (20% HAEC-CM). We observed that the HCECs cultured in the 20% HAEC-CM had an increased proliferative capacity, higher colony-forming efficiency (CFE), fewer apoptotic cells, and similar cell-junction formation capabilities and pump functionality compared with primary HCECs. Compared with CEM and 20% HAEC-Me, the 20% HAEC-CM system enhanced the functional properties of HCECs by reducing the generation of reactive oxygen species (ROS), maintaining the membrane potential (Δψm) at higher levels, reducing the expression of the p53 protein, and increasing the level of survivin protein expression. This study may shed light on the expansion of HCECs and the clinical applications of these cells in regenerative medicine, especially in corneal tissue engineering.

Use of silicone tubes to repair canalicular lacerations via a novel method.

PURPOSE: To develop a novel method to repair canalicular lacerations using silicone tubes. METHODS: A total of 47 adult patients (47 eyes) with canalicular lacerations were collected from the outpatient department from November 2010 to December 2012. The age ranged from 16 to 53 years. Among the 47 eyes, 37 had lower canalicular lacerations, 6 had upper canalicular lacerations, and 4 had bicanalicular lacerations. A soft probe was made using a stainless steel acupuncture needle, which was inserted into the lumen of the proximal part of the catheter to increase its rigidity. The probe was then inserted into the lacrimal sac and nasolacrimal duct. After retrieval of the catheters, the two ends of the silicone tube were securely tied (end to end) to the catheters. The silicon tube outside the nostril formed a U-shape. The catheters were then pulled upward until the silicone tube was completely located in the canalicular system. The catheters were cut off of the silicone tube near the site of the connection. The two ends of the silicone tube were cut short, -2mm out of the lacrimal punctum, and tied securely, end to end. The length of the tube between the upper and lower punctum was adjusted to ensure that no tension was present in the medial cathus, and the suture was removed through the nostril. The silicone tube was removed 3-10 months after this novel canalicular intubation procedure (NCI). RESULTS: All cases were anatomically rehabilitated after surgery. The silicone tube was removed after implanted in 3-10 months (mean 4.5 +/- 1.3 months), the average follow-up time was 11.8 months after removal. In total, 45 eyes in all 47 eyes (95.74%) were free from obstruction. Among them, 41 eyes (91.11%) achieved complete success (completely disappearance of epiphora after tube removal), 4 eyes (8.89%) achieved partial success (irritation occurs under stimulation conditions, such as wind or cold conditions), 4 eyes showed postoperative tearing, with three eyes having inferior lacrimal duct laceration, and one eye with superior canalicular laceration. Apart from two cases (4.26%) suffering inferior punctum splitting, no other associated issues occurred with the silicone tube or iatrogenic injury and lacrimal complications. CONCLUSION: For adult patients with canalicular laceration, the NCI was an effective, atraumatic surgery, which has fewer complications than traditional canalicular suture.

[Study on microsurgical procedures for pterygium operation and its therapeutic effects].

OBJECTIVE: To study the microsurgical procedures for the treatment of large primary pterygium and their therapeutic effects. To observe the recurrence rate, the changes of visual acuity after microsurgery and the pathological relationships between pterygium and cornea/sclera under the surgical microscope. METHODS: Forty-six eyes of forty-one patients with pterygium which invading the cornea over the pupil border were included. Pterygium was dissected by various methods under surgical microscope. The pathological relationship between the pterygium and cornea/sclera was observed. The lengths of the pterygium head and its three parts were measured. Degenerative Tenon's capsule was removed totally and the wound was covered by rotated conjunctival flaps. These patients were followed-up for 12.0 - 50.2 months (median: 22.4 months). Changes in visual acuity and recurrence rate after operation were observed. RESULTS: The average length of the total pterygium heads was (6.3 +/- 0.4) mm. The head was divided into three parts: the apical, loose and adhesive parts. The apical part was located at the top of the pterygium head with a length of (1.7 +/- 0.4) mm. The tissue of apical part was compact, hard, translucent and adhered to the cornea tissue. The adhesive part was a band in front of the anterior border of the limbus and paralleled to the limbus. The width of adhesive part was (0.9 +/- 0.1) mm and was tightly adhered to the cornea. The loose part lied between the apical and the adhesive part. The length of which was (3.6 +/- 0.4) mm and could be separated from the cornea easily. The neck and the body parts of pterygium could be separated easily from the limbus and sclera. Non-corrected visual acuity averaged 0.3 (ranged from finger count to 0.7) before the operation and averaged 0.7 (ranged from finger count to 1.5) 1 month postoperatively (Wilcoxon signed rank test u = 5.435, P < 0.01). Pterygium relapsed in 5 eyes with a recurrence rate of 11% (5/46). CONCLUSIONS: There is a regular pathological relationship between the pterygium and the cornea/sclera under surgical microscope, which is fundamental for the microsurgery of the pterygium. Extensively degenerated Tenon's capsule should be removed totally and the defect should be covered by rotated conjunctival flaps. The recurrence rate is low and the visual acuity increases significantly after the operation.

[Management of corneo-conjunctival malignant melanoma with "no touch technique" surgical excision and corneoscleral lamellar keratoplasty].

OBJECTIVE: To study the management of "no touch technique" surgical excision and corneoscleral lamellar keratoplasty in the treatment of corneo-conjunctival malignant melanoma. METHODS: Surgical excision for corneo-conjunctival malignant melanoma in six cases, from October 1989 to January 2004 in Zhongshan Ophthalmic Center, were performed. The entire tumors were removed in one piece without touching the tumor (no touch technique). The incision was outlined 4-6 mm outside the pigmented conjunctival mass, and 2 mm outside the corneal component. Dissecting deep enough without any tumor cell left to yield a tumor-free bed. After surgical incision, the host eyeball defect was covered with partial or total corneoscleral lamellar graft from fresh donor, and the conjunctiva defect was covered with or without amniotic membrane transplantation. All the patients were followed up to 6 months. RESULTS: The tumors in the remaining conjunctival margins were completely removed by local dissection combined with cryotherapy in the six cases. Three cases were treated with partial corneoscleral lamellar keratoplasty, two cases with total corneoscleral lamellar keratoplasty and one case with total corneoscleral lamellar keratoplasty plus amniotic membrane transplantation. All cases were subsequently treated with 0.01% thiotepa eyedrops and supplemental dacarbazine chemotherapy. At the average 43.5 months follow-up time (14.6 years to 6 months), no evidences of local recurrence of malignant melanoma or distant metastasis were observed. CONCLUSION: The clinical study suggests that using surgical excision of "no touch technique" to remove tumor in whole piece with corneoscleral lamellar keratoplasty is effective in the treatment of corneo-conjunctival malignant melanoma.