Background Human limbal allograft transplantation or limbal autograft transplantation are the primary approaches to the severe corneal-blindness,but their application in clinic were limited because of the defects of donor material.With the development of tissue engineering technology,transplantation of in vitro cultured limbal epithelial stem cells is being an advanced management.Objective The aim of this work was to expand human limbal epithelial stem cells ex vivo under the guidance of confocal microscope and to lay the foundation for fabricating ex vivo cultured cell sheets.Methods Ten eyes of ten patients were examined with the Heidelberg Retina Tomography Ⅲ Rostock Cornea Module(HRT3-RCM)to elucidate the structure of the human corneoscleral limbus and to correlate limbal epithelial dimensions.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank of Henan Eye Institute were cut into suitable explants.Then,this study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for limbal epithelial stem cell markers (p63,cytokeratinl9)and differentiation markers(keratin 3,involucrin).This experimental procedure was approved by the Ethic Committee of Henan Provincial People's Hospital.The written informed consent was obtained from subjects before initiation of any examination.Results The palisade morphology of human limbus was imaged clearly on the laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The cell-island phenomenon was seen in the basement membrane under the laser scanning in vivo confocal microscopy.The oblique sections of limbus showed many papilla-like epithelial columns below the superficial limbal epithelia.Throughout the experiment duration,the epithelial cells grew well with the migration rates from limbal tissue (68.62± 16.94)％ and the migration time(5.83 ±2.04)days,which depended on the tissue freshness.Compared with the second and forth batch of tissue,the migration rates of the third and sixth batch of tissues were significantly higher(P＜0.05),and the migration time was evidently longer in the forth and sixth batch of tissue compared with the first,second,third and fifth batch(P＜0.05).The positively expressing rates in the cultured corneal stem cells were 4.05％ and 36.52％ for p63,26.07％ and 40.55％ for CK19,57.88％ and 40.81％ for K3,64.66％ and 59.19％ for involucrin.Conclusion Human limbal epithelial stem cells can be successfully and purposefully obtained from the limbal tissue based on the guidance confocal miscroscope.The cultured corneal stem cells can grow well on the denuded amniotic membrane

Background Research demonstrated that mitochondrial pathway plays a key role in cell apoptosis.Purendan supermicropowder(PRD),a traditional Chinese medicine,may be a potentially effective therapy for neuron apoptosis in diabetic retina.Objective This study was carried out to investigate the effects of PRD on aldose reductase(AR)activity,neuron apoptosis and mitochondrial pathway in retina of diabetic rat.Methods Thirty-six clean male Wistar rats were randomly divided into normal control group,diabetes model group,PRD treatment group randomly and 12 rats for each group.The diabetes models were established by intraperitoneal injection of 25 mg/(kg · d)streptozotocin(STZ)for 3 consecutive days,and blood glucose ≥ 16.7 mmol/L was taken as the standard.PRD solution of 1.8 g/(kg · d)was lavaged in 12 models for 3 months.The eyeballs were enucleated for the preparation of retinal tissue homogenate and slice.AR activity in the retina was detected by ultraviolet spectrophotometry,and neuron apoptosis in retina was assayed by TUNEL staining.Western blot was used to assess the expressions of bcl-2,bax,cyt-c and caspase-3 protein in the retina.The use of animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Committee(Version 1988).Results Statistically significant differences were found in AR activity and AI among the normal control group,diabetic group and PRD groups(F=90.115,165.540,P＜0.01),and those of diabetic group were evidently higher than the normal control group and PRD group(P＜0.01,P＜0.01).The positive TUNEL cells mainly located in inner nuclear layer and retinal ganglion cell layer.The expressions of bax,cyt-c,caspase-3,bcl-2 and bcl-2/bax in retina were obviously different among these three groups(F =51.332,41.262,25.888,38.564,47.870,P＜0.01),and the expression of bax,cyt-c and caspase-3 protein in diabetic group evidently elevated in comparison with the normal control group and PRD group(t ＝ 10.32,11.04,6.91,P ＜ 0.01)and the expressions of bcl-2 protein and bcl-2/bax value were significantly lower in diabetic rats than in the normal control rats(t =18.05,12.23,P＜0.01).AR activity by AI of retina,the expressions of bax,cyt-c and caspase-3 proteins in retina were obviously lower in PRD group than in diabetes model rats(P ＜ 0.01),and the expression of bcl-2 protein and bcl-2/bax value were significantly higher in PRD group than in diabetes group(P＜0.01).Conclusions PRD can protect retina against the damage caused by high glucose by suppressing AR activity by downregulating the expressions of bax,cyt-c,caspase-3 proteins,increasing the expressions of bcl-2 protein in retina of diabetic rats and further inhibiting the mitochondrial pathway and reducing cell apoptosis in retina of diabetic rats.

Background Biomaterials for corneal tissue engineering must demonstrate several critical features for potentialutility invivo, includingtransparency, mechanicalintegrity, biocompatibilityand slow biodegradation. Silk film biomaterial had been characterized to meet these functional requirements. ObjectiveThis study was to investigate the feasibility of physico-crosslink regenerated silk fibroin film as tissue engineered corneal scaffold. MethodsHuman corneal epithelial cells(CECs) links were cultured by regular method and CECs in logarithmic phase were than incubated on physico-crosslink regenerated silk fibroin film membrane. The shape of cultured human CECs was observed after 24,48 and 72 hours under the inverted microscope and scanning electron microscope( SEM ) ,and the CECs were cultured on culture plates as controls. The growth state of CECs on regenerated silk fibroin film was observed daily for 7 days by MTT, and cell cycle analysis and the presence of apoptosis of human CECs were examined by flow cytometry after incubation on regenerated silk fibroin film. Regenerated silk fibroin filmCECs (4 mm×3 mm) were implanted into the corneal stroma of the right eyes of New Zealand white rabbits. At the end of 4 and 8 weeks after implantation, the appearance of the ocular surface was examined using slit lamp and corneal neovascular area was measured. Corneal histopathological examination was carried out to assess the degradation of graft materials and immunohistochemistry was performed to detect the expression of CD34 in the corneal tissue after operation. ResultsThe morphology and structure of CECs were identical using the two cultured Methods when observed under the inverted microscope and SEM after 24,48 and 72 hours. No significant difference was found in the A490 value 1,2,3,4,5,6 or 7 days after incubation on regenerated silk membrane and in culture plates ( Fmethod =0. 641 ,P＞0.05 ). The apoptosis rates of CECs on regenerated silk membrane or culture plates were 1.8％ and 2.0％ and the amount of cells in G2/G1 phase was 1. 956 and 1. 945, respectively. Histopathological examination showed that the regenerated silk membrane material degraded and was replaced by regular collagen tissue 2 months after implantation,and the presence of neovascular area and inflammatory cells were less prominent in 2 months than 1 month post-implantation. The expression level of CD34 in corneal tissue was evidently lower 1 and 2 months after operation than the Ad-VEGF165-induced positive control group (P＜0. 05), and no significant differences were seen when compared with normal CECs(P＞0.05). ConclusionsPhysico- crosslink regenerated silk fibroin film is an excellent biomaterial for tissue engineered corneal scaffold with good biocompatibility.

BackgroundPosterior capsule opacification(PCO) is the main cause inducing low vision after extacapsular cataract extraction. Our previous study determined that polylysine-ethylene diamine tetraacetic acid (EDTA) (PLE) can suppress the incidence of PCO. ObjectiveThe goal of this experiment was to investigate the inhibition of polylysine-EDTA on rabbit lens epithelial cells (LECs)proliferation in vitro and the effective concentrations of polylysine-EDTA. MethodsThe anterior capsular membranes from 10 3-month-old clean New Zealand white rabbits were digested and then cultured to obtain the LECs. The second and third generation of LECs were inoculated on the 96-hole culture plate with the cell density of the 1 × 105/ml. 12.5,25.0,50. 0,100. 0 μmol/Lof PLE were added into the culture medium for 48 hours respectively,and the DMSO medium was used at the same way as the control group. The proliferation of the LECs was then detected by MTT method and the inhibitory rate of PLE on LECs growth was calculated. ResultsLECs grew at a near normal state in ≤25.0 μmol/L PLE groups,however,cultured LECs were out of shape and the numbers decreased with the weakened adhesion ability in ≥50.0 μ mol/L PLE groups. The A490 values of LECs were 0. 278±0. 013,0. 266±0. 028,0. 260±0. 022 and 0. 247±0. 012 in 12. 5,25.0,50. 0, 100. 0 μmol/L polylysine-EDTA groups respectively and were lower than 0. 311 ±0. 038 of DMSO control group( P=0. 035,0. 011,0. 009,0.013 ). The inhibitory rates of 12. 5,25.0,50. 0, 100.0 μmoL/L PLE on LECs proliferation were 10.61％ , 14.47％ , 16.40％ and 20. 58％ respectively. ConclusionsPolylysine-EDTA can inhibit the growth and proliferation of LECs in vitro at a dose-dependent manner.

BackgroundThe quest to look for seed cells is a hot spot of cornea transplant research in solving the problem of the lack of donor. Bone marrow mesenchymal stem cells(BMSCs) have been successfully induced into retinal ganglion cells(RGCs) in vivo,but the successful induction of BMSCs into corneal endothelial cells has not been reported.Objective This experiment was to study the transplantation of BMSCs on corneal endothelial surface using the splitting Descemet's membrane. MethodsFour healthy adult rhesus monkeys were divided into the experimental group ( 3 monkeys) and control group ( 1 monkey). Mesenchymal stem cells (MSCs) were isolated from bone marrow by density gradient centrifugation combined with adhering means. The cultured cells were identified by flow cytometry and its ability to differentiate was determined by allowing them to differentiate into adipocytes in vitro and labeled by 5-bromodeoxyuridine ( BrdU ) for subsequent identification. Corneal grafts of 7 mm in size with tearing of the Descemet' s membrane were prepared in the experimental group and control group. After labeling by 5-bromodeoxyuridine( BrdU ) ,cultured cells were transplanted onto the endothelial surface of cornea grafts in the experimental group, but no cultured cells were seeded in the graft of the control group. The corneal grafts were then sutured in situ, and were removed 1,2 or 3 months after operation to examine the distribution and connection between transplanted cells and their morphologic changes under the electron microscope. Results High purity MSCs were harvested by density gradient centrifugation combined with adhering method. Cultured cells reached confluency after 12 to 16 days, presenting with a spindle shape and parallel or swirling arrangement. Flow cytometry analysis showed that 94.26％ of cells were positive for CD29,7. 51％ for CD34 and 4. 02％ for CD45. Larger nuclei filled with plastosomes, golgiosomes and rough endoplasmic reticula were found on the graft under the transmission electron microscope( TEM ). After 3 weeks, MSCs were differentiated into adipocytes where Oil Red O staining resulted in an orange-red staining in the cytoplasm and blue staining in the nuclei. The transplanted cells attached loosely on the endothelial surface of the corneal graft and came in contact with each other in one month. The shape of the cells appeared as spindle-shaped and polygonal after 2 months and became tightly packed after 3 months. The positive cells retained the BrdU label and presented with brown nuclei. No endothelia cells grew in the cornea graft in the control group, with an absence of BrdU labeling. Conclusions Mesenchymal stem cells can be transplanted onto the corneal endothelial surface successfully and form a monolayer using the centrifugation method, and present with good survival and proliferation ability.

Retinopathy of prematurity( ROP),a leading cause of childhood visual impairment,is a vascular proliferative retinopathy in premature infants. Although the hallmark of ROP is abnormal retinal vasculature, a growing body of research in animals and observations in patients support a critical role for the neural retina in the ROP disease process. ROP can affect the foundation of the retina and optical nerve during the development duration in childhood, presenting with abnormalities in electroretinogram and multifocal electroretinogram. In addition,it was found that ROP has different degrees of influence on the development of the dioptric system ,which will result in refractive error as the child progress to adulthood. The relationship of the electrophysiology features or refractive abnormality and ROP was summarized in this article.

BackgroundDiabetic retinopathy (DR) is a progressive vision-threatening complication of diabetes mellitus,but its pathogenic mechanism is still unclear. Recent studies showed that it may be associated with the inflammation response of retinal capillary. Cytokines can cause induction of proinflammatory and adhesion molecules and thereby increase monocyte endothelial cell adhesion, which is now accepted as the early key event in the development of DR. ObjectiveThe present study was to determine the relationship between the stages of DR and the levels of serum vascular endothelial growth factor (VEGF) , interleukin-2 ( IL-2 ), tumor necrosis factor-alpha (TNF-α) in diabetic patients. Methods This was a pilot case-controlled study. Ninety patients with type 2 diabetes mellitus were included in this clinical trial and 30 healthy individuals were enrolled as controls. The patients were grouped into the non-diabetic retinopathy(NDR) group,background DR group and proliferative DR(PDR) group according to the results from ophthalmoscopic examination and fundus fluorescein angiography(FFA) ,with 30 patients for each group. The levels of serum VEGF,IL-2,TNF-α were assayed by ELISA and compared among the 4 groups.Written informed consent was obtained from each subject before received any related medical examination to this study. ResultsThe mean serum VEGF levels were(217.35±27. 87)ng/L,(298.31±49.26)ng/L,and(341.23±40. 18)ng/L, respectively, and mean serum IL-2 levels were( 12. 12± 1. 57 )ng/L, (16.43 ±2. 26 )ng/L, and (21.36±0. 86) ng/L,respectively and mean serum TNF-α levels were( 11.63±0. 94) ng/L, ( 17. 52±0. 65) ng/L,and(22. 01±0. 87 ) ng/L respectively in the patients with NDR ,background DR and PDR, showing significant differences from healthy controls with( 193.46±37. 39 ) ng/L for serum VEGF, ( 8. 99 ±0. 57 ) ng/L for serum IL-2 and ( 7.31 ±0. 52 ) ng/L for serum TNF-α ( F =126. 38, P＜0. 0 1 ;F =120. 37, P＜0. 01 ;F =99. 84, P＜0. 01 ). The levels of serum VEGF, IL-2, and TNF-α in the patients with the NDR,background DR and PDR were increased significantly. The level of serum VEGF showed the positively significant correlation with serum IL-2 level and TNF-α level ( r =0. 749, P ＜ 0.01 ; r =0. 631,P＜0. 01 ). The serum levels of VEGF, IL-2 and TNF-α showed a significantly positive correlation with the prolongation and severity of DR(r=0. 791 ,P＜0. 01 ;r=0. 665 ,P＜0. 01 ;r=0. 632,P＜0. 01 ). ConclusionsVEGF, IL-2 and TNF-α play active roles in the generation and development of diabetic retinopathy, and the level of serum VEGF is closely associated with the levels of serum IL-2 and TNF-α. during the development of DR.

BackgroundIt is important to measure the corneal curvature, anterior chamber depth (ACD) and axial length accurately for calculating IOL power. The interchange outcomes from different measuring methods and apparatus will cause unreliable IOL power. ObjectiveThe present study was to compare the differences of corneal curvature, anterior chamber depth (ACD) measured by IOLMaster and Orbscan Ⅱbefore and after laser in situ keratomileusis(LASIK) and further compare the axial length measured by IOLMaster and A-ultrasound. Methods One hundred and thirty eyes from 65 consecutive myopic patients before LASIK and 56 eyes of 28 cases with 1-month follow-up duration after LASIK in Henan Eye Institute were enrolled in this study. The K value, ACD between IOLMaster and Orbscan Ⅱ as well as results of axial length between IOLMaster and A-ultrasound were compared by using paired t test. The agreements of the measured values among IOLMaster, Orbscan Ⅱ and A-ultrasound were evaluated using Bland-Altman plot. ResultsBefore LASIK,the K value measured by IOLMaster,Orbscan Ⅱ were ( 43.32 ± 1.52 ) D and ( 42.99 ± 1.45 ) D respectively with the difference value of( 0. 33 ±0. 03 ) D, showing a significant difference(t=10. 380,P=0.000) and a positive relation between them(r=0.971,P=0.000). After LASIK,the K value measured by IOLMaster, Orbscan Ⅱwere(39. 02±2. 14) D and ( 38.91 ±2. 04) D with the difference value (0. 12±0. 33 ) D, presenting a significant differences between them (t =2.715, P =0.009). Bland-Altman plots indicated the disagreement in K value and uninterchangeable. Before LASIK, the ACD measured by IOLMaster,Orbscan Ⅱ and A-ultrasound were ( 3.72 ± 0. 22 ) mm, ( 3.69 ±0. 22 ) mm and ( 3.75± 0.27 )mm respectively and no significant differences were found between them (P ＞ 0. 05 ). Axial length measured by IOLMaster significantly prolonged in comparison with A-ultrasound(25.59± 1. 01 mm vs 25.22±0.99 mm ) , and the difference was( -0. 37 ±0. 30 ) mm, showing significant difference ( t =- 14. 098, P =0. 000 ) and positive correlation ( r =0. 954, P =0. 000 ). Axial length values measured by IOLMaster were ( 25.54 ± 1.05 ) mm in preoperation and ( 25.48 ± 1.01 ) mm in postoperation with the difference (0.052±0. 412)mm, showing statistically insignificant difference between them (t=0. 946,P=0. 348). ConclusionsKeratometries measured by IOLMaster,Orbscan Ⅱ are much more different. Therefore,these two methods are not recommended to use interchangely. ACD measured by IOLMaster,Orbscan Ⅱ and A ultrasound are proved to obtain the similar results and is clinically interchange. Axial length measured by IOLMaster is longer than that measured by A-ultrasound.

BackgroundIron-containing foreign body trapped in the eyeball wall without affecting the opticus occurs occasionally in clinic. Operation always is performed in an attempt to avoid the deposition of rust in different tissues of the eye-balls. However,a few animal experimental studies showed that a small foreign body does not affect the retina and opticus in the period of three months. The question of whether surgery needs to be carried out is worth discussion. ObjectiveThe aim of this study was to evaluate the influence of posterior intrascleral iron foreign body on the rabbit retina and opticus. MethodsTwelve healthy adult Japan flap-eared white rabbits were randomly divided into two groups. Medium carbon iron with rust or without rust( size of 2. 0 mm × 1. 0 mm×0. 2 mm) were implanted into the posterior sclera of the left eye to create the animal model with iron foreign body in the eyeball wall. The cornea, anterior chamber, crystalline lens, vitreous and fundus of the rabbits were observed under a slit lamp microscope 1weekbeforeoperationand 1week, 2weeks, 1monthand 3months after operation.Flash electroretinogram(F-ERG) and flash visual evoked potential (F-VEP) were recorded at the time points mentioned above. All the rabbits were sacrificed and the eye balls were extracted at the end of the experiment, and the position of the iron foreign body was determined. The histopathological examinations of the retina and opticus were performed under the light microscope. This experiment complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. ResultsThere were no statistical differences for the a-wave amplitude of F-ERG among different time points( F =1. 885,P =0. 129 ) and different treatment groups ( F =1. 188, P =0. 340 ), as with the ERG b-wave amplitude ( time: F =2. 73, P =0. 064 ; group : F =1. 114, P =0. 367). The differences in the latencies of F-VEP N1-wave were insignificant among the different time points( F =1. 605, P =0. 263 ) as well as various groups ( F=1. 556, P =0.314 ), and those of F-VEP P1 -wave were not evidently changed ( time: F =2. 329, P =0. 092 ; group : F =2. 186, P =0. 103 ). No correlations were seen between the time factor and grouping factor ( P ＞ 0. 05 ). There was no apparent siderosis bulbi change during the follow-up duration. No morphological abnormality in the retina and optical nerve was found under the light microscope. At the end of the experiment,intrascleral iron foreign body was wrapped by surrounding tissue in a stable condition. Conclusions The small posterior intrascleral iron foreign body, whether it is oxidized or not, does not produce distinctive functional or pathological damage on retina and opticus in the short term.

BackgroundOptic nerve injury lead to apoptosis of retinal ganglion cells ( RGCs ), and its mechanism of apoptosis is endoplasmic reticulum stress (ERS). So, decreasing of ERS may protect the injury of RGCs. ObjectiveThe present study was to investigate the mechanisms of endoplasmic reticulum stress(ERS) and the protective effects of human umbilical cord blood stem cells on partial optic nerve crush injury. MethodsThe optical nerves were crushed with a 40 g clip by holding for 60 seconds to establish the partial optical nerve injury model in the left eyes of 102 SPF SD rats,and 10 μl of mRNA and 10 μl of nerve growth factor were injected into the vitreous immediately after the establishment of the model. The morphological changes of retinal ganglion cells(RGCs) were examined under the light microscope after 3,7,14,21 and 28 days and the RGCs number was calculated. The apoptosis rates of RGCs were detected by the TUNEL technique after 3, 12,24,45,72 hours and 1 week. The expression levels of GRP78 mRNA and CHOP mRNA were detected by reverse transcription polymerase chain reation (RT-PCR). This procedure followed the Regulations for the Administration of Affair Concerning Experimental Animals by Hunan Provincial Science and Technology Committee.Results The number of RGCs was significantly decreased with the prolongation of time of optical nerve injury in the model injury group,whereas the number of RGCs in the human cord blood cells group was reduced at a slower rate( Ftime =20. 100,P =0. 007 ). At various time points after the injection of human cord blood cells, the survival of RGCs was evidently increased in comparison with the model group(P＜0. 01 ). The apoptosis rate of RGCs was considerably elevated with injury time prolongation both in the model group and human cord blood cells group,but no apoptosis was seen from 3-24 hours after operation,and only a small amount of apoptotic cells were found in the human cord blood cells group from 48 hours through 1 week than in the model group(P＜0. 01 ). In the human cord blood cells group,GRP78 mRNA level was significantly higher and the CHOP mRNA level was significantly lower than those in the injury group at identical time points(P＜0. 01 ). ConclusionsIn the rat optic nerve partial crush model,ERS induces the apoptosis of RGCs. Human umbilical cord blood stem cells can protect RGCs from ERS injury by inhibiting apoptosis.