Corneal endothelial cell density and morphology in Chinese patients with pseudoexfoliation syndrome

AIM: To evaluate the corneal endothelial cell density and morphology in Chinese patients with pseudoexfoliation syndrome (PEX). METHODS: Medical records of 16 patients (20 eyes) with PEX who presented to our institution between July 2008 and June 2010 were retrospectively reviewed. Thirteen eyes had combined glaucoma. The information of five apparently normal fellow eyes in these patients was also recorded. Left eyes of 20 patients with bilateral senile cataracts but no other eye disease were included as controls. Specular microscopy was performed in all eyes to analyze for corneal endothelial cell density and morphology. Cell density, coefficient of variation in cell size, and percentage of hexagonal cells in corneal endothelium were evaluated. RESULTS: The mean corneal endothelial cell density in the PEX eyes was 2298+/- 239 cells/mm(2), significantly lower than that in the cataract eyes (2652+/- 18 cells/mm(2), P=0.026), but there were no significant differences in coefficient of variation of cell size and frequency of hexagonality between these two groups. No significant differences in the three parameters were found between the apparently normal fellow eyes and the PEX eyes or the cataract eyes, or between the PEX eyes with and without glaucoma. CONCLUSION: Corneal endothelial cell density may decrease in Chinese patients with PEX. The development of glaucoma in PEX eyes does not seem to be related with the change in corneal endothelial cell density or morphology.

Corneal endothelial cell density and morphology in low and moderate myopic Chinese eyes

AIM:To describe and compare the corneal endothelial cell density and morphology in young,low and moderate myopic Chinese adults in Malaysian Chinese population.·METHODS:Non-contact specular microscopy(Topcon SP3000P,Tokyo,Japan) was performed in low(n =78;21.22±1.51 years) and moderate(n =78;21.82±1.40 years)myopic subjects.The mean of three consecutive measurements of endothelial cell density(MCD),coefficient of variation(CV) in the cell size,and hexagonal appearance of the cell were obtained.·RESULTS:In low myopic eyes the MCD was 3 063.0±176.2/mm2,the mean CV was 33.4 ±4.0% and the mean hexagonal appearance of the cell was 57.9 ±2.7%.In moderate myopic eyes the MCD was 2961.6 ±159.0/mm2,the mean CV was 33.9 ±3.6% and mean hexagonal appearance of the cell was 56.2 ±4.7%.There were statistically significant differences in MCD(P 【0.000) and hexagonal appearance of the cell(P 【0.005) between low and moderate myopic eyes.·CONCLUSION:The corneal endothelial cell layer in more myopic eyes tends to have less MCD and cell hexagonality compared to lower myopic eyes.Nevertheless,there is no significant difference in CV between low and moderate myopic eyes.

Corneal endothelial cell density and morphology and central corneal thickness in Guangxi Maonan and Han adolescent students of China

AIM: To investigate the corneal endothelial cell density and morphology and central corneal thickness in the Guangxi Maonan and Han adolescent students of China.METHODS: Noncontact specular microscope(Topcon SP3000 P, Tokyo, Japan) was performed in 133 adolescent students of Maonan nationality(M:F 54:79)and 105 adolescent students of Han nationality(M:F 50:55),5 to 20 y of age, who were randomly selected from 3schools in Huanjiang Maonan Autonomous County of Guangxi Zhuang Autonomous Region of China.Parameters studied included endothelial cell density,mean cell area, coefficient of variation in cell size,percentage hexagonality and central corneal thickness. RESULTS: Endothelial cell density, mean cell area,coefficient of variation in cell size, percentage hexagonality and central corneal thickness in the study population were(2969.50 ±253.93) cells/mm2,(339.23 ±29.44) μm2,(29.96 ±4.07) %,(64.58 ±9.41) % and(523.71 ±32.82) μm in Maonan and(2998.26 ±262.65) cells/mm2,(336.11±30.07) μm2,(29.89±5.03) %,(64.91±11.64) % and(524.39 ±33.15) μm in Han, respectively. No significant differences were observed in endothelial cell density,mean cell area, coefficient of variation in cell size,percentage hexagonality and central corneal thickness between Maonan and Han(P =0.615, 0.659, 0.528, 0.551,0.999). In Maonan and Han, we found age was negatively correlated with endothelial cell density and percentagehexagonality and positively correlated with mean cell area and coefficient of variation in cell size. Negative correlation was also found between central corneal thickness and age in Han, whereas no correlation was found in Maonan. CONCLUSION: There were no differences between Maonan and Han in corneal endothelial cell density and morphology and central corneal thickness. In these two nationalities, there were statistically significant decrease in endothelial cell density and percentage hexagonality with increasing age and statistically significant increase in cell area and coefficient of variation in cell size with increasing age. Central corneal thinned with increasing age in Han, whereas difference did not attain statistical significance in Maonan.

Manifestations of type 2 diabetes in corneal endothelial cell density,corneal thickness and intraocular pressure

We sought to evaluate central corneal thickness（CCT）,corneal endothelial cell density（ECD）and intraocular pressure（IOP）in patients with type 2 diabetes mellitus（DM）and to associate potential differences with diabetes duration and treatment modality in a prospective,randomized study.We measured ECD,CCT and IOP of125 patients with type 2 DM（mean age 57.1±11.5 years）and compared them with 90 age-matched controls.Measured parameters were analyzed for association with diabetes duration and glucose control modalities（insulin injection or oral medication）while controlling for age.In the diabetic group,the mean ECD（2511±252 cells/mm^2）,mean CCT（539.7±33.6μm）and mean IOP（18.3±2.5 mmHg）varied significantly from those the control group[ECD：2713±132 cells/mm^2（P〈0.0001）,CCT：525.0±45.3μm（P=0.003）and IOP：16.7±1.8 mmHg（P〈0.0001）].ECD was significantly reduced by about 32 cell/mm^2 for diabetics with duration of〉10 years when compared with those with duration of〈10 years（P〈0.05）.CCT was thicker and IOP was higher for diabetics with duration of〉10 years than those with duration of〈10 years（P〉0.05）.None of the measured parameters was significantly associated with diabetes duration and treatment modality（P〉0.05）.In conclusion,subjects with type 2DM exhibit significant changes in ECD,IOP and CCT,which,however,are not correlated with disease duration or if the patients receive on insulin injection or oral medications.

Efficacy of ICL combined with Trans-PRK on corneal endothelial cell density in patients with high myopia

Objective:To explore the efficacy of implantable collamer lense (ICL) combined with laser keratectomy via corneal epithelium Trans-PRK in the treatment of patients with high myopia and its effect on corneal endothelial cell density.Methods: A prospective analysis was performed on patients with ultra-high myopia admitted to the department of ophthalmology of our hospital from March 2014 to March 2017. Six months after ICL implantation, the patients still had myopia and received Trans PRK treatment again. The number of cases was 30 and 52 eyes. Patients were followed up postoperatively. Uncorrected visual acuity (UCVA), best corrected visual acuity (BCVA), dioptre (D), intraocular pressure, ICL arch height, anterior chamber volume, anterior chamber depth, and changes in corneal endothelial cell counts were observed preoperatively, 6 months after ICL implantation and 6 months after Trans PRK so as to assess the effectiveness and safety of the surgery.Results: UCVA and BCVA of the eyes were significantly higher 6 months after ICL implantation and 6 months after Trans-PRK than those before the surgery, and UCVA of the eyes 6 months after Trans-PRK was significantly higher than that 6 months after ICL implantation. There was no increase in intraocular pressure in all patients after surgery. There was no significant difference in intraocular pressure preoperatively, 6 months after ICL implantation and 6 months after Trans PRK. The ICL arch height of the eyes 6 months after Trans-PRK was slightly lower than that 6 months after ICL implantation, but the difference was not statistically significant. Both anterior chamber volume and anterior chamber depth were significantly lower 6 months after ICL implantation and 6 months after Trans PRK than whose before the surgery, and anterior chamber depth 6 months after Trans PRK was significantly lower than that 6 months after ICL implantation. The number of corneal endothelial cells 6 months after ICL implantation was slightly less than that before surgery, but it was not statistically significant. The number of corneal endothelial cells 6 months after Trans PRK was higher than that 6 months after ICL implantation and close to the preoperative level.Conclusion: Trans PRK can effectively improve the eye diopter of patients with high myopia and residual myopia after ICL implantation. It has little effect on the density of corneal endothelial cells, can significantly improve visual acuity, and achieve the expected operation effect with safe operation, which is worthy of clinical promotion.

Corneal endothelial cells and acoustic cavitation in phacoemulsification

Postoperative complications of phacoemulsification,such as corneal edema caused by human corneal endothelial cell(CEC)injury,are still a matter of concern.Although several factors are known to cause CEC damage,the influence of ultrasound on the formation of free radicals during surgery should be considered.Ultrasound in aqueous humor induces cavitation and promotes the formation of hydroxyl radicals or reactive oxygen species(ROS).ROS-induced apoptosis and autophagy in phacoemulsification have been suggested to significantly promote CEC injury.CEC cannot regenerate after injury,and measures must be taken to prevent the loss of CEC after phacoemulsification or other CEC injuries.Antioxidants can reduce the oxidative stress injury of CEC during phacoemulsification.Evidence from rabbit eye studies shows that ascorbic acid infusion during operation or local application of ascorbic acid during phacoemulsification has a protective effect by scavenging free radicals or reducing oxidative stress.Both in experiments and clinical practice,hydrogen dissolved in the irrigating solution can also prevent CEC damage during phacoemulsification surgery.Astaxanthin(AST)can inhibit oxidative damage,thereby protecting different cells from most pathological conditions,such as myocardial cells,luteinized granulosa cells of the ovary,umbilical vascular endothelial cells,and human retina pigment epithelium cell line(ARPE-19).However,existing research has not focused on the application of AST to prevent oxidative stress during phacoemulsification,and the related mechanisms need to be studied.The Rho related helical coil kinase inhibitor Y-27632 can inhibit CEC apoptosis after phacoemulsification.Rigorous experiments are required to confirm whether its effect is realized through improving the ROS clearance ability of CEC.

Cytotoxic effects of betaxolol on healthy corneal endothelial cells both in vitro and in vivo

AIM: To demonstrate the cytotoxic effect of betaxolol and its underlying mechanism on human corneal endothelial cells(HCE cells) in vitro and cat corneal endothelial cells(CCE cells) in vivo,providing experimental basis for safety anti-glaucoma drug usage in clinic of ophthalmology. ·METHODS: In vivo and in vitro experiments were conducted to explore whether and how betaxolol participates in corneal endothelial cell injury. The in vitro morphology,growth status,plasma membrane permeability,DNA fragmentation,and ultrastructure of HCE cells treated with 0.021875-0.28g/L betaxolol were examined by light microscope,3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide(MTT) assay,acridine orange(AO)/ethidium bromide(EB) double-fluorescent staining,DNA agarose gel electrophoresis,and transmission electron microscope(TEM). The in vivo density,morphology,and ultrastructure of CCE cells,corneal thickness,and eye pressure of cat eyes treated with 0.28g/L betaxolol were investigated by specular microscopy,applanation tonometer,alizarin red staining,scanning electron microscope(SEM),and TEM. · RESULTS: Exposure to betaxolol at doses from 0.0875g/L to 2.8g/L induced morphological and ultrastructural changes of in vitro cultured HCE cells such as cytoplasmic vacuolation,cellular shrinkage,structural disorganization,chromatin condensation,and apoptotic body appearance. Simultaneously,betaxolol elevated plasma membrane permeability and induced DNA fragmentation of these cells in a dose-dependent manner in AO/EB staining. Furthermore,betaxolol at adose of 2.8g/L also induced decrease of density of CCE cells in vivo,and non-hexagonal and shrunk apoptotic cells were also found in betaxolol-treated cat corneal endothelia. ·CONCLUSION: Betaxolol has significant cytotoxicity on HCE cells in vitro by inducing apoptosis of these cells,and induced apoptosis of CCE cells in vivo as well. The findings help provide new insight into the apoptosis-inducing effect of anti-glaucoma drugs in eye clinic.

Visual acuity and endothelial cell density with respect to the graft thickness in Descemet's stripping automated endothelial keratoplasty: one year results

AIM: To evaluate the visual acuity and endothelial cell density according to the thickness in Descemet’s stripping automated endothelial keratoplasty(DSAEK)one year after surgery.METHODS: DSAEK patients’ data were reviewed. Thirty seven eyes of 37 patients who underwent DSAEK for pseudophakic bullous keratopathy(PBK) were included in this study. Graft thickness was measured with optical coherence tomography(OCT) 12 mo after DSAEK. Eyes were divided into 3 groups based on the graft thickness:thick(】200 μm), medium-thick(150-200 μm) and thin(【150 μm). Best corrected visual acuity(BCVA),endothelial cells density(ECD) and complications were assessed and comparisons were done between groups.RESULTS: Median thickness of postoperative grafts was 188(range 73-317 μm). There was no significant difference in age, sex, preoperative BCVA, or follow-up period between DSAEK groups. At postoperative 12 mo,mean BCVA was 0.28±0.10 in thick graft group, 0.52±0.08 in medium-thick graft group, and 0.72 ±0.06 in thin graft group. Thin grafts showed better postoperative BCVA as compared with the medium-thick and thick grafts(P =0.001). Thick graft group had 1637.44 ±88.19-mm2,medium thick graft had 1764.50±34.28-mm2 and thin graft group had 1845.30 ±65.62-mm2. Thin graft group had better ECD at 12 mo after surgery(P =0.001).CONCLUSION: Thin grafts after DSAEK ensure better visual rehabilitation. Eyes with thin grafts had significantly lesser loss of ECD compared to eyes withmedium-thick and thick grafts one year after surgery.

Nerve Growth Factor Modulate Proliferation of Cultured Rabbit Corneal Endothelial Cells and Epithelial Cells

Summary： In order to investigate the effect of nerve growth factor （NGF） on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF.MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mI. and 500 U/mI. NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

AIM:To investigate the morphological altering effect of transforming growth factor-β2(TGF-β2) on untransfected human corneal endothelial cells(HCECs)in vitro.METHODS:After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology,cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy,immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2(9 μg/L) altered HCE cell morphology after treatment for 36 h, increased the mean optical density(P 【0.01) and the length of F-actin,reduced the mean optical density(P 【0.01) of the collagen type IV in extracellular matrix(ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72 h.·CONCLUTION: TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

Effect of recombinant human platelet-derived growth factor B on cat corneal endothelial cell viability mediated by adeno-associated virus

AIM: To transduce recombinant human platelet-derived growth factor B (PDGF-B) gene adeno-associated virus(AAV) to in vitro cultured cat corneal endothelial cell (CEC) and observe the effect of the expressed PDGF-BB protein on the viability of cat CEC. METHODS: Cat cornea endothelium was torn under microscope and rapidly cultivated in DMEM to form single layer CEC and the passage 2 endothelial cells were used in this study. The recombinant human PDGF-B gene AAV was constructed and transduced into cat CEC directly. Three groups were as following: blank control group, AAV control group and recombinant AAV group. At 24 hours, 48 hours, and 5 days after transduction, total RNA was extracted from the CEC by Trizol and the expression of PDGF-B gene was detected by fluorescence quantitative polymerase chain reaction. Viability of the transduced CEC was detected at 48 hours after transduction by MTT assay. Cell morphology was observed under inverted phase contrast microscope. RESULTS: With the torn endothelium culture technique, we rapidly got single layer cat CEC. At 24 hours, 48 hours and 5 days after transduction, fluorescence quantitative polymerase chain reaction showed there was no significant difference of the expressed PDGF-B gene mRNA between blank control group and AAV control group (P>0.05). In contrast, there were significant differences between two control groups and recombinant AAV group (P<0.05). MTT assay showed that in recombinant AAV group, the expressed PDGF-BB protein could promote the viability of cat CEC. Morphology observation showed at 48 hours after transduction, cells in CEC-AAV-PDGF-B group proliferated into bigger scales in regular triangle to hexagon shape with distinct boundary, while the number of cells was significantly less in the two control groups. CONCLUSION: The recombinant AAV-PDGF-B expresses biological active PDGF-BB protein in cat CEC, which promotes the viability and proliferation of cells.

Effect of basic fibroblast growth factor on cat corneal endothelial cell proliferation

AIM: To investigate the function of basic fibroblast growth factor (bFGF) on cat corneal endothelial cells proliferation. METHODS: Cat corneal endothelial cells were primarily cultured, stimulated with bFGF for different period, the proliferation of cells was assayed by modified tertrozalium salt (MTT) method, and the morphologic changes were observed with inverted phase contrast microscope and transmission electron microscope. RESULTS: At 1, 3 and 5 days after bFGF was added to cat corneal endothelial cells, the result of MTT in 490nm showed significant difference than that in control group, and the difference was most significant in 10ng/mL group. CONCLUSION: bFGF can promote proliferation of cat corneal endothelial cells. 10ng/mL is the relatively most effective dose.

The study of human PDGF-B gene transferred to cat corneal endothelial cells

AIM: To demonstrate that human platelet-derived growth factor-B (PDGF-B) cDNA could be Expressed in primary cultured cat corneal endothelia cells by using gene transfer techniques; to explore a useful tool for the further studies of the molecular mechanisms of corneal endothelium failure and provide a potential effective genetic therapy for the blind patients. ' METHODS: Human PDGF-B cDNA was isolated from human placent by RT-PCR and inserted into pcDNA(4) vector to construct recombinant eukaryotic expression plasmid pcDNA(4)-PDGF-B. The full length was confirmed by the DNA sequencing analysis. By tearing endothelium technique we obtained pure single layer of cat corneal endothelial cells. The pcDNA(4)-PDGF-B eukaryotic Expression vector was transferred into cat corneal endothelial cells by Effectene (TM) lipofectine. The transfection efficiency of Effectene (TM) lipofectine in pcDNA(4)-B was detected with pcDNA(4)-GFP. 5 days later, RT-PCR was used to check the PDGF-B expression. Cell viability was tested by modified tertrozalium salt (MU) method. Cell morphology was observed under inverted phase contrast microscope. RESULTS: The human PDGF-B cDNA was isolated successfully from healthy parturien placent tissue and the sequence was confirmed by computer automatic sequence and PCR analysis. Pure single layer cat corneal endothelial cells were successfully cultured by tearing endothelium technique. Effectene (TM) lipofectine transfection technique could be effectively used to transfer pcDNA(4)-PDGF-B into cat corneal endothelial cells in vitro, the transfection efficiency was 30%. RT-PCR result showed that human PDGF-B gene was highly expressed in transfected cat corneal endothelial cells. The expressed PDGF-BB protein promoted the viability of cat corneal endothelial cells. CONCLUSION: Human platelet-derived growth factor-B (PDGF-B) cDNA could be highly Expressed in cultured cat corneal endothelial cells by gene transfection techniques. Expressed PDGF-BB protein significantly promoted the viability of cat corneal endothelial cells, thus provided a potential effective method for corneal endothelium blindness genetic therapy.

Changes in endothelial cell density following penetrating keratoplasty and deep anterior lamellar keratoplasty

· AIM: To compare the corneal endothelial cell density (ECD) of clear grafts after penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK). · METHODS: The study included 44 and 54 patients treated with PK and DALK, respectively, between March 2006 and April 2010. Corneal ECD was examined using specular microscopy at postoperative 1, 3, 6, 12, and 18 months, and the values were compared. · RESULTS: Corneal ECD reduction in the PK group was 7.4%, 15.2%, 23.5%, and 28.9% at 3, 6, 12 and 18 months respectively after surgery, compared with 4.2 % in the first month (P <0.01). These figures were 3.0%, 6.7%, 7.2%, and 7.7% at 3, 6, 12 and 18 months respectively, compared with 2.2 % in the first month in the DALK group (P >0.05). · CONCLUSION: Compared with DALK,PK significantly reduced ECD of the clear grafts. These results suggest that survival of endothelial cells in grafts is better after DALK than after PK. ·

Effect of p27Kip1 Inhibition on Proliferation of Bovine Corneal Endothelial Cells by RNA Interference

Three plasmids （pGenesil-P1, pGenesil-P2, pGenesil-P3） with different p27Kip1-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endothelial cells （bCEC） were investigated. Plasmid expressing irrelevant shRNA with a random combination was used as negative control （pGenesil-HK）. The recombination of four plamids was confirmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kip1 mRNA and p27Kip1 protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group （non-transfected group）, pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The proliferation rates of the pGenesil-P3 group, the pGenesil-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was detected by flow cytometry （FCM）. Compared with the control groups, the proliferation rate of the pGenesil-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kip1 could down-regulate the expression of p27Kip1 effectively and increase the proliferation of bCEC. RNA interference （RNAi） may be an effective means to promote the proliferation of CEC.

Mechanism of induction of fibroblast to corneal endothelial cell

Objective:To explore mechanism of nduction of fibroblast to corneal endothelial cell.Methods:Rabbit conjunctiva fibroblasts were used as feeder cells,rabbit oral mucosa epithelial cells were used as seed cells,and human denuded amniotic membrane was used as carrier to establish tissue engineering corneal endothelium.The transformation effect was observed.Results:As concentration of mitomycin C increased,cell survival rale gradually decreased,cell proliferation was obviously inhibited when concentration ≥25μg/mL;5 days alter being treated by 5μg/mL.mitomycin C,cell body was enlarged and extended without cell fusion,however after being treated by 0.5 μg/mL mitomycin C,cell body was significantly proliferated and gradually fused:alter 3 weeks of culture,stratified epithelium appeared on rabbit oral mucosa epithelial cells,differentiation layers were 4-5 and were well differentiated,the morphology was similar to corneal endothelial cells;Under electron microscope,surface layer of cells were polygonal,tightly connected to another with microvilli on the bonier,there was hemidesmosome between basal cells and human denuded amniotic membrane.Conclusions:Fibroblast cells have the potential of multi-directional differentiation,effective induction can promote emergence of intercellular desmosomes between seed cells and emergence ol epithelial surface microvilli,and differentiate to the corneal endothelial cell.However,clinical application still needs more research and safetv evaluation.

A study of chitosan blend film as a carrier of corneal endothelial cells

In order to evaluate the cytocompatibility and biocompatibility of a new kind of chitosan blend film as a carrier of corneal endothelial cell, rabbit corneal endothelial cells cultured in vitro were breeded onto the film. After a cell monolayer formed, the scanning electron micrography was performed. After inplanted into anterior chamber, slit lamp observation, thickness metering, specular microscopy and HE staining were performed at random time after operation to evaluate the biocompatibility. Inflmmnation in anterior, thickness of cornea, cell density, hexagonality and cell size of the surgical cornea were taken as the indexes of biocompatibility. The cultured cells exhibited a confluent monolayer 10 days after incubation, which proved the satisfactory cytocompatibility of this film. Biocompatibility assay results suggested the implantation feasibility of the film as a carder of corneal endothelial cells.

High-density lipoprotein endocytosis in endothelial cells

AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

Effects of Intraocular Lens Implantation without Viscoelastic Agents on Corneal Endothelial Cells

Objective: To determine the protective effects of intraocular lens implantation without viscoelastic agents on corneal endothelial cells. Methods: Patients with age-related cataract were randomly divided into two groups: Group A (24 patients, 30 eyes) underwent phacoemulsification and intraocular lens implantation without viscoelastic agents, and group B (21 patients, 30 eyes) underwent phacoemulsification and intraocular lens implantation with viscoelastic agents. The corneal endothelial cell counts, percentages of hexagonal cells, and central corneal thicknesses were evaluated at 1 week, 1 month, and 6 months after surgery. Results: There was no significant difference in preoperative basic characteristics between the two groups (p > 0.05). The postoperative corneal endothelial cell count and percentage of hexagonal cells in both groups decreased compared with preoperative values at 1 week, 1 month, and 6 months after surgery, and the decrease of group A was significantly lower than that of group B at all time points (p p > 0.05). Conclusions: Compared with regular intraocular lens implantation, intraocular lens implantation without viscoelastic agents is less damaging to corneal endothelial cells, resulting in greater corneal safety.

Mechanism of 5-Fluorouracil-Induced Apoptosis on Cultured Corneal Endothelial Cells

Repeated subconjunctival injections with 5-fluorouacil (5-FU) after trabeculectomy are widely used in glaucoma patients for the inhibition of excess scar formation in wound site. The aim of this study was to evaluate the toxic effects of 5-FU and mechanisms of drug-induced apoptosis in cultured porcine corneal endothelial cells. Cellular damage and the caspase pathway were estimated with a MTT assay. The apoptotic characteristics were detected with flow cytometry, a TUNEL test and Western blotting. The results indicated that 5-FU was toxic to corneal endothelial cells in a time- and dose-dependent manner. Pretreatment with a general caspase inhibitor, Z-VAD-FMK, a caspase-8 inhibitor, Z-IETD-FMK, and a caspase-9 inhibitor, Z-LEHD-FMK, reversed 5-FU-induced cellular damage. Following exposure to 5-FU, a flow cytometric assay with MitoLight dye demon-strated the loss of mitochondrial membrane potential. A positive TUNEL test revealed that cellular DNA apoptosis occurred following exposure to 0.05, 0.1, and 0.5 mg/ml 5-FU for 15 h. Annexin V-FITC and negative propidium iodide (PI) staining indicated that the cell membrane underwent apoptosis upon exposure to 0.1 and 0.5 mg/ml 5-FU for 15 h. The Western blot assay demonstrated up-regulation of the Bax, p53 and p21 proteins induced by 5-FU. Taken together, these data reveal that 5-FU-induced cellular apoptosis in corneal endothelial cells may be mediated through caspase-8, caspase-9 and mitochondrial regulated pathways, as well as by up-regulation of Bax-, p53-, and p21-dependent signal transduction pathways.