Background Studies have determined that nucleotide binding oligomerization domain 2 (NOD2) plays a key role in innate immune response.However,whether NOD2 participates in the nature defense of fungal keratitis is unclear.Objective This study was to investigate the expression and significance of NOD2 on cornea in the initial of Aspergillus fumigatus keratitis (AFK) in rats.Methods Seventy-two adult clean Wistar rats were randomized into the normal control group,only corneal epithelial scraped group and AFK model group,and the AFK models were established by incubating Aspergillus fumigatus to cornea after corneal epithelium was scraped.All the operations were performed in the right eyes of rats.Reverse transcription PCR (RT-PCR) was carried out to detect the expression of NOD2 mRNA in corneal epithelium 4,8,16,24 hours after operation.Twenty-four hours after operation,the expression of NOD2 protein in rat corneas was examined by immunochemistry and immnunofluorescence technology.Also,the rat corneas were obtained for regular histopathological examination.The use and care of the animals complied with Institutional Animal Care and Use Committee Guidebook by NIH.Results All the models were made successfully.RT-PCR revealed that a fewer NOD2 mRNA were expressed on cornea in the normal control group,but the expressing levels of NOD2 mRNA were increased in the only corneal epithelial scraped group and AFK model group.Compared with only corneal epithelial scraped group,the elevated values of NOD2 mRNA expression in the AFK model group were statistically significant at 4,8,16 and 24 hours after operation (t =-0.409,-0.439,-0.534,-0.618,all at P=0.000).The histopathological examination displayed that the cornel tissue had intact structure in the normal control group,and partly corneal epithelial deficiency,slight corneal swelling and fewer neutrophil granulocytes were seen in the only corneal epithelial scraped group.However,corneal ulcer,severe corneal edema and a lot of neutrophil granulocytes were exhibited in the AFK model group.Immunochemistry and immnunofluorescence staining evidenced that weaker expression of NOD2 was visualized in the corneal epithelial and endothelial layers,and obviously enhanced staining was seen in the AFK model group.The expressing levels (absorbancy) were 0.045 ± 0.005,0.050 ± 0.005 and 0.092 ± 0.006 in the normal control group,only corneal epithelial scraped group and AFK model group,respectively,showing a significant increase in the AFK model group compared with the only corneal epithelial scraped group (t =0.042,P =0.000).Conclusions Expression of NOD2 is upregulated in the corneas with AFK,suggesting that NOD2 participates the natural defense in the initial of fungal keratitis.NOD2 may play an important role in the process of anti-fungal innate immune response in cornea.

Background To maintain corneal transparency is important for good visual function.A new treatment concept and the selection of surgical techniques and timing of surgery are critical for stopping the infringement of cornea tissue after alkali burning and other chemical warfare agents.Objective This study was to investigative the ultrastructure and histopathological status following the femtosecond laser-assisted deep lamellar keratoplasty (DLK) for acute alkali burn of cornea.Methods Acute corneal alkali burn models were established in 12 New Zealand rabbits by putting the 6 mm filter paper with 1 mol/L NaOH at the central cornea for 30 seconds.The rabbits were randomly allocated to femtosecond laser-assisted DLK group and model control group according to the randomized number table method.Femtosecond laser-assisted DLK was performed to transplant the corneal grafts of domestic rabbits to the model rabbits 24 hours after burning.The rabbits were sacrificed 1 week,2 weeks and 4 weeks after modeling,and the corneas were extracted for the preparation of corneal section.The cornea were performed with hematoxylin and eosin staining to assess the histopathological status under the optical microscope,and the ultrastructure of grafts and corneas was examined under the transmission electron microscope (TEM).Results Acute corneal alkali bourn models were successfully eatablished.In the fourth week after surgery,corneal graft was clear in the femtosecond laser-assisted DLK group.However,corneal swelling,conjunctival congestion and neovascularization were found in the model control group.Histopathological examination revealed the defect of corneal epithelium,edema of stroma,loose arrangement of collagen fibers,much vacuoles,few neovascularization and infiltration of a large number of inflammatory cells in the model control group,but in the femtosecond laser-assisted DLK group,the inflammatory response was slight.More desmosomes among the endothelial cells were seen,and the nuclei were intact in the grafts.In the fourth week after surgery,the transplanted corneas were transparent with the regular arrangement of collagen fibers and entire fibroblasts in the femtosecond laser-assisted DLK group under the TEM.However,flat surface corneal epithelial cells and shedding of some epithelial cells were exhibited in the modelcontrol group.Conclusions Femtosecond laser-assisted DLK can effectively alleviate the inflammatory response,promote epithelial healing and enhance intercellular tight junction in the cornea with acute alkali burn.

Background Corneal blindness is one of the major blinding eye diseases in China.With the development and progress of tissue engineering technology,tissue-engineered corneas offers a new approach to the treatment of corneal diseases.To select and cultivate ideal seed cells is a foundation of construction of tissueengineered corneas.Objective This study was to evaluate the efficiency of stripe off the Descemet membrane with endothelium plus enzymic digestion in the isolation of corneal endothelial cells and analyze the bionomics of rabbit corneal endothelial cells (CECs) in vitro.Methods Descemet membrane was stripped from fresh cornea of New Zealand rabbit under the dissection microscope.Descemet membrane with endothelium was incubated in trypsin and EDTA solution at 37 ℃ and then purified for CECs subculture in vitro.The morphology of the cultured cells was observed under the inverted microscope and marked by CM-Dil dye solution.Then the shape of the cells was observed by hematoxylin and eosin staining and the cells were identified for the expression of neuron specific enolase (NSE) using immunochemistry.The viability of the cells were evaluated by trypan blue staining.The surface structure of the cells were examined under the scanning electron microscope.Intercellular zonula occludens-1 (ZO-1) was identified by immunofluorecsence staining.Results A large number of purified CECs were obtained from Descemet membrane with endothelium through enzymic digestion.Cultured cells grew well and formed monolayer 5-7 days later with the cobblestone stone-like arrangement.The survival rate of the cells was 95％.CECs presented with the red annular fluorescence for CM-Dil with the labeling rate ＞90％.NSE was positively expressed in the cytoplasm.Polygon CECs were seen by hematoxylin and eosin staining and showed the brown staining.Abundant microvilli on the cellular surface and interconnected foot process were seen under the scanning electron microscope.ZO-1 showed the green fluorescence.Conclusions The method of striping off the corneal Descemet membrane with endothelium plus enzymic digestion can obtain abundant CECs.Cultured cells have good biological properties.This study may offer a feasible application in the engineering of corneal transplant membrane.

Background Histopathological examination is an important approach to the basic and clinical study in ophthalmology.Different fix methods of retinal samples produce a large impact.The first fixed way often is used at home,and the last fixed way is preferred abroad.The comparative study between the two ways is lack.Objective This study was to compare rat retinal frozen sections following 4 different fix methods and confirm a simpler and better way.Methods Forty eyeballs were extracted from 40 SD rats and were randomly divided into four groups using random number table,ten for each group.The eyeballs were firstly fixed in 4％ paraformaldehyde overnight,4％ paraformaldehyde for 2-3 hours,formalin-acetic acid-alcohol (FAA) mixture solution,and then OCT embedding was performed in 3 groups.However,the samples were fixed in cold acetone following first liquid nitrogen frozen,OCT embeded in the forth group.Retinal serial sections of 4 groups were examined and compared after hematoxylin and eosin staining under the optical microscope.Results Retinal tissue was loosened interlayerly,and clustering of cells in the inner nuclear layer (INL) and outer nuclear layer (ONL) were seen in the 4％ paraformaldehyde overnight fix group.The arrangement of interlayer and cells was improved in the 4％ paraformaldehyde for 2-3 hours group compared with 4％ paraformaldehyde overnight fix group.The retinal structure was closer among the layers in the FAA fixed group.In the later cold acetone fixed group,retinal morphology was more clear with the intact structure and regularly arranged cells in the ONL,INL and ganglion cell layer(GCL),as an living histology structure.Conclusions The first fix of retina produces a large impact on retinal morphology and structure.However,cold acetone fixation following first liquid nitrogen frozen is simpler,less time-consuming and more efficient way for the histopathological examination of retina.

Background The malignant degree of the diffuse large B-cell lymphoma (DLBCL) is high.Radiation therapy is sensitive,but the therapeutic schedule can not be unified,reasonable and effective therapeutic schedule is important in clinic treatment of the DLBCL.Objective This study was to access the effect of X-ray on the cell cycle and cell apoptosis of the DLBCL.Methods DLBCL cell line was cultured and the cell suspension was inoculated to 12-well cultured plate.The cells exposed to X-ray for 6,12,24,48 hours with the irradiation doses of 2,4,6,8 Gy respectively.The cell proportions of different cycles were assayed by flow cytometer,and cell apoptosis index was evaluated and calculated.Non-irradiation cells served as controls.Results The cells grew well with a good cell vitality and round-like shape,and the growth was stable in generation 2 or 3.With the increase of X-ray doses and the lapse of irradiation time,the cell proportions in G0-G1 phase gradually reduced (Fdose =45.58,P=0.00; Ftime =43.11,P=0.00).However,cell proportions in G2-M phase were gradually increased as the increase of X-ray doses and lapse of irradiation time (Fdose =40.77,P =0.00 ; Ftime =91.91,P =0.00).In addition,the apoptotic proportion of the cells was significantly elevated with the increase of X-ray dose and irradiation time (Fdose =87.36,P =0.00; Ftime =37.31,P =0.00).Conclusions X-ray irradiation retards DLBCL in the G2-M phase and causes cell apoptosis in dose-and time-dependent manner.

Background Intracranial vascular malformation causes different neurophthalmological symptoms and signs due to oppressing visual-related tissue and cranial nerves after hemorrhage.However,there is no availably systematic clinical research on this disease up to now.Objective This retrospective cases analysis was to explore the neurophthalmological features of intracranial vascular malformation.Methods The clinical datas were collected from 100 cases with intracranial vascular malformation in Affiliated Third Hospital of Sun Yat-sen University from June,2007 to June,2013.The neurophthalmological clinical features including general condition,initial symptom,concomitant symptoms and image results were retrospectively surveyed.Results Neurophthalmological symptoms and signs were found in 20 patients with intracranial vascular malformation by CT and MRI with the detectability 20％ (20/100).The main neurophthalmological symptoms were visual field defect (50％,10/20) and vision loss (45％,9/20).The neurophthalmological signs were pupil abnormalities (35％,7/20).Other neurophthalmological features included optic nerve atrophy (5 ％,1/20),papilloedema (5 ％,1/20) and oculomotor nerve palsy (5 ％,1/20),etc.The relevant cranial diseases included cerebral arteriovenous malformation in 60％ patients (12/20),cavernous angiomas in 35％ (7/20) and venous malformation with cavernous angiomas (1/20).The lesions of cerebral arteriovenous malformation were located in occipital lobe (4 cases),parietal-occipital area (3 cases),temporal lobe (3 cases) and frontal lobe (1 case),midbrain area (1 case) ; while those of cavernous angiomas were located in parietal lobe (1 case),occipital lobe (1 case),gyrus cingulated area occipital lobe (1 case),carvenous sinus (1 case),temporal lobe (1 case),parietal-temporal area (1 case) and pons area (1 case).Intracranial hemorrhage occurred in 14 patients in subarachnoid space (6 cases) and brain (8 cases).Conclusions The patients with intracranial vascular malformation have different neurophthalmological symptoms and signs.Sufficient attention should be paid to the patients with neurophthalmological symptoms and signs.

Background Evidence-based medicine (EBM) has been widely applied to solve clinical issues internationally,however,the awareness of oculists in China still is dissatisfied,and the relevant issue survey can offer the training guidance.Objective This study was to survey the status of the cognition and practice of EBM in ophthalmologists in China and analyze the problems in their EBM practice.Methods Two hundred and thirty-three ophthalmologists were enrolled in the 18th Congress of Chinese Ophthalmological Society in August,2013.A survey about EBM was performed by self-made questionnaire,including general data from the surveyors,the way and channel of understanding EBM,the attitude toward EBM,clinical practice of EBM and influencing factors.The educational background,professional qualifications,ranked distribution of hospitals which surveyors came from,size of the citys and attribute of hospitals were stratified and analyzed.SPSS 17.0 software was used for statistical analysis.Results A total of 233 questionnaires were distributed,and 233 were regained with the response rate 100％,and 214 valid questionnaires were included,with the effective rate 91.8％.50.5％ of the respondents (104/206) knew EBM in university.The proportion of those believe EBM initially as medical students was 41.8％,and 49.1％ (102/208) were aware of EBM in recent 8 years.And 47.7％ (101/211) believed that EBM should be given priority in clinic,and 7.5％ (16/211) were indifferent to EBM.Only 3.8％ (8/211) surveyors thought EBM to be little important.The proportion of the respondents choosing ‘ know’ or ‘ moderately-know’ were 69.1％ (148/214),35.0％ (75/ 213),21.5％ (46/213) and 57.4％ (121/211) respectively in EBM,Meta analysis,Cochrane as well as Preferred Practice Patterns (PPPs) with a higher receiving degree in well-educated individuals (P =0.000,0.000,0.001,0.000).EBM was well practiced in 11.7％ (25/211) oculists,with a higher using frequency in senior ophthalmologists,and 17.8％ (89/495,frequency) oculists consulted the EBM literature to resolve problems in clinic,with a significant difference in the using frequency of EBM among the different educational background subjects (x2=13.406,P=0.037).The top three barriers which affected the application of EBM in clinical practices were lack of time and the limited energy,feeling it difficult to evaluate the evidence correctly as well as the absence of cases proving the efficacy to treat based EBM.The type of EBM evidence mostly used by respondents was medical database (36.7％),and CNKI (33.5％) and PubMed (38.4％) were the mostly used Chinese and English database,respectively.Respondents who have never searched the Chinese or English database occupied up to 7.3％ and 17.2％,respectively.Conclusions The cognition degree and applying frequency are still lower in ophthalmologists in China.The training of EBM should be strengthened in medical students.The guidance measurement should be taken to encourage eye doctors to apply EBM.

Background Randomized controlled trial (RCT) can offer high quality of evidence for the medical decisions.Consolidated standards of reporting trials (CONSORT) statement,an international criteria for RCT report,is a guideline and methodology of RCT report.However,the assessment and application of RCT according to CONSORT are dissatisfactory in China.Objective This study was to evaluate the reporting quality of ophthalmic diseases related RCTs published in SCI journals from mainland China and offer a good methodology of RCT report.Methods The RCT literature were searched on PubMed database according to Ophthalmology, Eye and all of their inferior subjective terms,and the published papers were limited in mainland China and before March,2013.Included papers were evaluated independently and checked by two searchers.The methodology and results of all retrieved papers were stratifiedly analyzed by the year of CONSORT published and revised.Results Sixty eligible RCTs were assessed,and the intervention methods were primarily surgery trials (34/60).Forty-four papers presented the comparison study of two groups with the median sample size of 76.0.Only 22％ (13/60) trial reports gave the clear identification of RCT in the title.Majority of papers described trial design,eligibility criteria for participants,detail of interventions and outcome measures.Twelve pieces of articles reported how sample size was determined,and 41 pieces referred settings and locations,but only 6 pieces recorded interim analyses and stopping guidelines.After CONSORT 2010 Statement was published,the proportion of trial reports that included details of the randomization had little improvement (RR=1.06,95％ CI:0.62-1.82).Only 42％ trial reports (25/60) offered a description about blinding method.Conclusions RCT reports related to ophthalmic diseases from mainland China in SCI journals are still flawed with certain shortcomings.Following the guidance of the CONSORT statement can improve the quality of ophthalmic diseases related RCT from mainland China.

Background Insulin can promote the occurrence of myopia.It has been proven that insulin receptor exists in human retinal pigment epithelial (RPE) cells and can promote RPE cells to secrete transforming growth factor-β2(TGF-β2),which is one of the most important myopic signal molecules.Objective This study was to investigate if PI3K/Akt mediates the promotive effects of insulin on proliferation of human RPE cells and secretion of TGF-β2.Methods Human RPE cell line,ARPE-19 cells,were regularly cultured using DMEM containing 10％ fetal bovine serum,and 10× 103 U/ml insulin,LY294002,10× 103 U/ml insulin+LY294002,Wortmanin,10× 103 U/ml insulin+Wortmanin were added into the medium respectively for 48 hours,and the regularly cultured cells served as blank controls.The proliferation value (absorbance,A) of the cells was evaluated by MTS,and the TGF-β2 level in the cell supernatant was detected by ELISA.The relative expression of TGF-β2 mRNA in the cells was assayed using reverse transcription PCR (RT-PCT) 1 hour and 2 hours after the addition of reagents.Results MTS showed that the proliferation value of the cells in the insulin+LY294002 group was 0.75±0.03,which was significantly lower than that in the insulin group (0.98± 0.04).No significant difference was seen in the proliferative value between the insulin+Wortmanin group and the insulin group (0.97±0.07 versus 0.98± 0.04,P＞0.05).ELISA revealed that the content of TGF-β2 in the the cell supernatant was (11.59±2.85) pg/ml and (49.16± 10.94) pg/ml in the insulin + LY294002 group and the insulin + Wortmanin group,respectively,showing a significant decline in comparison with (548.50±35.18) pg/ml in the insulin group (both at P＜0.05).A significant difference was found in the TGF-β2 content between the insulin+LY294002 group and the insulin+Wortmanin group (t =8.131,P =0.000).The RT-PCR showed that 1 hour and 2 hours after addition of the reagents,the expression levels of TGF-β2 mRNA in the cells were lower in both insulin+LY294002 group and insulin+Wortmanin group than those in the insulin group (P＜0.05).The decline range of TGF-β2 mRNA expression level was more significant in the insulin+LY294002 group than that in the insulin+Wortmanin group at 1 hour (t=4.176,P=0.014) rather than at 2 hours (t=0.756,P=0.492).Conclusions Insulin can promote the proliferation of human RPE cells and secretion of TGF-β2 through PI3K/Akt pathway.This may be one of the mechanisms of insulin causes myopia.

Background Researchers are paying increasing attention to the effect of cellular senescence in vascular dysfunctional diseases,and it is hypothesized that cellular senescence may also be involved in the development of diabetes related vascular complications.The outstanding feature of cellular senescence is the upregulation of beta-galactosidase.Objective This study was to investigate the effects of high glucose on cell senescent in vitro and in vivo on bovine retinal endothelial cells (BRVECs) and mouse retina.Methods BRVECs were cultured and passaged,and the seventh generation of cells were employed in this study.The cells were divided into the control group and the high glucose culture group and cultivated using M199 medium containing 5.5 mmol/L or 25.0 mmol/L glucose,respectively.5-bromine-chlorine-4-3-indole-beta D galactose glucoside (X-Gal) staining was used to examine the expression of beta-galactosidase in the cells.Diabetic models were established in the SPF male C57BL/6J mice aged 8-10 weeks by intraperitoneal injection of streptozotocin (STZ),and the age-and gendermatched normal mice served as controls.The mouse retinas were collected and starched in the 48-well plates 3 months later.X-Gal staining was employed to calculate the positive cells.Results BRVECs grew well 24 hours after culture but showed irregular arrangement.Forty-eight hours later,the cells reached confluence with a tight connect.The ratios of positive BRVECs and total cells were (51.4±5.4) ％ and (36.6-±3.8) ％ in the high glucose culture group and the control group,with a significant difference between the two groups (t =-3.204,P =0.033).The number of positive cells for X-Gal in mouse retinas was (94.0± 15.1) /field in the diabetic group,which was higher than that in the control group ([60.0 ± 5.7]/field) (t =-2.974,P =0.041).Conclusions High glucose environment accelerates senescence of retinal cells,and high glucose induces premature cell senescence,which likely plays an important role in the pathogenesis of diabetic retinopathy.