Comprehensive Analysis of circRNA-Associated-ceRNA Networks in Human Corneal Endothelial Dysfunction.

PURPOSE: Circular RNAs (circRNAs) are a novel class of endogenous noncoding RNAs that regulate gene expression through the competitive endogenous RNA (ceRNA) mechanism. CircRNA-associated-ceRNA networks are closely related to oxidative stress-related diseases. Oxidative stress-induced dysfunction of the corneal endothelium (CE) is a major pathological feature in many corneal diseases. This study was aimed to analyze circRNA-associated-ceRNA networks in oxidative stress-induced CE dysfunction. METHODS: A CE dysfunction model was established using human corneal endothelial cells (HCECs) treated with H 2 O 2 at a concentration of 250 µM for 4 hours at 37°C. High-throughput sequencing was conducted to determine the expression profiles of circRNA, miRNA, and mRNA. Bioinformatic analyses, including Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes analysis, were conducted to identify the potential biological modules and pathologic pathways of dysregulated circRNAs. CircRNA-associated-ceRNA networks were established based on the data of sequencing and bioinformatic analyses. RESULTS: We obtained 108 differentially expressed circRNAs, including 77 upregulated and 31 downregulated circRNAs. GO analysis suggested that dysregulated circRNAs were mainly targeted to protein quality control for misfolded or incompletely synthesized proteins (biologic process), nuclear chromatin (cellular component), and ubiquitin protein ligase binding (molecular function). GO terms related to CE functions responding to oxidative stress were also identified. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that dysregulated circRNAs were mostly enriched in the adherens junction pathway. Network analysis identified several potential therapeutic targets for CE dysfunction. CONCLUSIONS: CircRNAs are significantly dysregulated in HCECs under oxidative stress. The circRNA-associated-ceRNA networks are closely related to HCEC functions. Targeting these networks might provide novel therapies for CE dysfunction.

Reconstruction of the ocular surface by autologous transplantation of rabbit adipose tissue-derived stem cells on amniotic membrane.

BACKGROUND: Corneal disease is the second most common cause of blindness in China. Clinically, treatment options for corneal diseases with limbal stem cell deficiency (LSCD) are limited due to a shortage of organ donors and inevitable immune rejection. This study aims to determine the efficacy of reconstructing the ocular surface using autologous cultivated adipose tissue-derived stem cells (ADSCs) and to develop a new clinical treatment for patients with LSCD. METHODS: A rabbit LSCD model was first established. Two weeks later, the animals were divided into three groups, including the sham group, the amniotic membrane transplantation group, and the ADSC combined with amniotic membrane transplantation group, and underwent surgery. The efficacy of reconstructing the ocular surface using ADSCs was evaluated using immunofluorescent staining, confocal microscopy (CM) observation, H&E staining, immunohistochemical staining, and scanning transmission electron microscopy observation one, two and four weeks after surgery. RESULTS: Evaluations of immunofluorescent staining of the cornea pre- and post-surgery yielded significantly lower scores for the corneas in the ADSCs transplantation group than for those in the sham group (F=-7, P=0.002, <0.05) and the amniotic membrane transplantation group (F=-4.67, P=0.018, <0.05) two weeks after surgery. Four weeks after surgery, the corneas of the ADSC combined with amniotic membrane transplantation group were scored significantly lower than those in the sham group (F=-8, P=0.007, <0.05) and the amniotic membrane transplantation group (F=-5.33, P=0.046, <0.05). The data suggest that the use of ADSCs to treat LSCD showed greater efficacy than the other treatment methods. The growth of ADSCs on the corneal surface was examined using confocal and electron microscopes. K3/K12 expression in the corneal epithelium, which was reconstructed by ADSCs, was negative, as shown by immunohistochemical staining. CONCLUSIONS: Ocular surface reconstruction can be improved by using ADSCs as seed cells and the amniotic membrane as a carrier, thus providing a new therapeutic strategy for patients with LSCD.

FTO regulates ocular angiogenesis via m6A-YTHDF2-dependent mechanism.

Pathological ocular angiogenesis commonly results in visual impairment or even blindness. Unveiling the mechanisms of pathological angiogenesis is critical to identify the regulators and develop effective targeted therapies. Here, we used corneal neovascularization (CNV) model to investigate the mechanism of pathological ocular angiogenesis. We show that N6-methyladenosine (m6A) mRNA demethylation mediated by fat mass- and obesity-associated protein (FTO) could regulate endothelial cell (EC) function and pathological angiogenesis during CNV. FTO levels are increased in neovascularized corneas and ECs under pathological conditions. In vitro silencing of FTO in ECs results in reduced cellular proliferation, migration, and tube formation under both basal and pathological conditions. Furthermore, FTO silencing attenuates suture-induced CNV in vivo. Mechanically, FTO silencing in ECs could increase m6A methylation levels in critical pro-angiogenic genes, such as FAK, leading to decreased RNA stability and increased RNA decay through m6A reader YTHDF2. Our study demonstrates that FTO regulates pathological ocular angiogenesis by controlling EC function in an m6A-YTHDF2-dependent manner.

CHA2DS2-Vasc score and CHA2DS2-Vasc-HS score are poor predictors of in-stent restenosis among patients with coronary drug-eluting stents.

OBJECTIVE: To evaluate the ability of two scoring systems (CHA2DS2-VASc score and CHA2DS2-VASc+hyperlipidaemia+smoking [CHA2DS2-VASc-HS score]) to predict in-stent restenosis (ISR) among patients undergoing drug-eluting stent (DES) implantation. METHODS: This retrospective study enrolled patients who underwent coronary angiography to assess coronary artery disease severity secondary to a diagnosis of stable angina or acute coronary syndrome that subsequently underwent DES implantations. Demographic, clinical, angiographic and biochemical parameters were compared between those patients that experienced ISR and those that did not during the study follow-up period. Univariate and multivariate logistic regression analyses were used to evaluate associations between the baseline parameters, the two scoring systems and ISR risk. RESULTS: A total of 358 patients (non-ISR group n = 316; ISR group n = 42) participated in the study. Compared with the non-ISR group, more patients in the ISR group had diabetes mellitus and received stents with smaller diameters but longer lengths. There were no significant differences with regard the predictive ability for ISR of either the CHA2DS2-Vasc or the CHA2DS2-Vasc-HS scores. Multivariate logistic regression analyses demonstrated that stent diameter, follow-up duration and glycosylated haemoglobin were independent risk factors for ISR. CONCLUSIONS: The CHA2DS2-Vasc and CHA2DS2-Vasc-HS scores did not predict ISR in patients after coronary DES placement.

Ileal perforation caused by a fish bone shortly after drug-eluting stent implantation for acute myocardial infarction.

During the past century, the incidence of myocardial infarction has markedly increased worldwide. Percutaneous coronary intervention with stent implantation is often considered as the first-choice treatment, especially in emergency cases. Current guidelines recommend delayed elective noncardiac surgery for such vulnerable patients. However, few suggestions are available regarding the exact treatment strategy for patients who have already undergone percutaneous coronary intervention but suddenly need emergent noncardiac surgery for an unrelated reason. We herein present a case involving a patient with acute myocardial infarction who had undergone implantation of a drug-eluting stent and developed an ileal perforation due to fish bone ingestion 3 days postoperatively. After carefully balancing the risks of stent thrombosis and uncontrollable bleeding, dual antiplatelet therapy and low-molecular-weight heparin were given with close monitoring. Emergency laparotomy and partial small bowel resection surgery were then performed, after which the patient eventually recovered. This case indicates a possible management strategy for patients with acute myocardial infarction complicated by emergency noncardiac surgery.

SIRT1-dependent AMPK pathway in the protection of estrogen against ischemic brain injury.

AIMS: Stroke is a major cause of mortality and disability, especially for postmenopausal women. In view of the protective action of estrogen, hormone therapy remains the only effective way to limit this risk. The objective of this study was to investigate the efficiency and underlying mechanisms of estrogen neuroprotection. METHODS: Subcutaneous injection of 17ß-estradiol in rats after ovariectomy (OVX) was used to manipulate estrogen level and explore the effects of estrogen in cerebral ischemic damage both in vivo and in vitro. Silent mating type information regulation 2 homolog 1 (SIRT1) knockout mice and adenosine monophosphate (AMP)-activated kinase (AMPK) inhibitor Compound C were also used to investigate the underlying pathway of estrogen. RESULTS: Estrogen deficiency induced by OVX aggravated brain infarction in experimentally induced cerebral ischemia rats, whereas estrogen pretreatment reduced ischemia-induced cerebral injuries. Neurons of estrogen deficiency models were susceptible to apoptosis under oxygen-glucose deprivation (OGD). In contrast, neurons with estrogen-supplemented serum exhibited restored resistance to cell apoptosis. In OGD neurons, estrogen promoted AMPK activation through estrogen receptor α, and neuroprotection of estrogen was prevented by AMPK inhibition. Estrogen increased SIRT1 expression and activation, and estrogen-induced AMPK activation disappeared in SIRT1 knockout neurons. Moreover, estrogen-induced neuroprotection was abolished in SIRT1 knockout mice and AMPK-inhibited rats. CONCLUSION: Our data support that estrogen protects against ischemic stroke through preventing neuron death via the SIRT1-dependent AMPK pathway.

Involvement of arterial baroreflex and nicotinic acetylcholine receptor α7 subunit pathway in the protection of metformin against stroke in stroke-prone spontaneously hypertensive rats.

Stroke is a leading cause of mortality and disability worldwide. There is growing evidence that metformin (Met) has potent neuroprotective effects; however, its mechanisms remain unclear. We examined the role of the arterial baroreflex and cholinergic-α7 nicotinic acetylcholine receptor (α7nAChR) anti-inflammory pathway in the beneficial effects of Met against stroke. Stroke-prone spontaneously hypertensive rats (SHRSP) were used to observe stroke development indicated by lifespan of SHRSP and the ischemic injury induced by permanent middle cerebral artery occlusion (MCAO). Sinoaortic denervation was used to inactivate the arterial baroreflex. MCAO were also performed in α7nAChR knockout (KO) mice. Briefly, Met increased the life span of SHRSP and reduced the infarct area induced by MCAO. Met also improved the function of arterial baroreflex. The beneficial effects of Met on stroke were markedly attenuated by blunting the arterial baroreflex. Met up-regulated the expression of vesicular acetylcholine transporter (VAChT) and α7nAChR, down-regulated the level of pro-inflammtory cytokines in serum and peri-infarct of ischemic brain. Arterial baroreflex dysfunction decreased the expression of VAchT and α7nAChR, showed upward tendency in the level of pro-inflammtory cytokines. Most importantly, arterial baroreflex dysfunction nearly abolished such effect of Met on cholinergic signaling. In addition, the α7nAChR KO mice also had significantly worse ischemic damage induced by MCAO, and neuroprotection of Met disappeared in α7nAChR KO mice. In conclusion, Met improved the arterial baroreflex function, and then enhancing cholinergic anti-inflammatory pathway in an α7nAChR-dependent manner, thereby effectively prevent ischemic induced brain injury and delayed stroke onset in SHRSP.

Dronedarone and Amiodarone Induce Dyslipidemia and Thyroid Dysfunction in Rats.

BACKGROUND/AIMS: Amiodarone, a thyroid hormone-like molecule, can induce dyslipidemia and thyroid dysfunction. However, the effects of dronedarone on lipid metabolism and of both dronedarone and amiodarone on thyroid function and lipid metabolism remain unknown. METHODS: Fifty male Sprague-Dawley rats were randomly divided into 5 groups (10 in each group): normal control (NC), amiodarone-treated (AMT), dronedarone-treated (DRT), rats treated with amiodarone combined with polyene phosphatidylcholine (AC), and rats treated with dronedarone combined with polyene phosphatidylcholine (DC). Rats were given amiodarone (120 mg/kg/d), dronedarone (120 mg/kg/d), and polyene phosphatidylcholine (200 mg/kg/d) for 13 weeks. At the end of weeks 4, 8, 12, and 13, plasma-free triiodothyronine (FT3), free thyroxine (FT4), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) were determined. At the end of this protocol, rats were sacrificed and the thyroid glands were isolated, weighed, and examined histopathologically. The protein expression of Bcl-2 was measured by immunochemical staining. The mRNA expression of thyroglobulin (Tg), type-1 deiodinase (D1), and thyroid peroxidase (TPO) were detected by polymerase chain reaction (PCR). RESULTS: Compared with the NC group, FT3 and FT4 levels in the DRT and DC groups significantly increased at week 4 but declined thereafter. The AMT and AC groups had lower FT3 levels but comparable FT4 levels. The levels of TG, LDL-c, and HDL-c in the NC group were lower than those in the other groups whereas the LDL-c/HDL-c ratio was lowest in the AMT group. Bcl-2 expression significantly increased in the DRT group. The mRNA expression of Tg increased whereas the mRNA expression of D1 decreased. Dronedarone induced hyperthyroidism at the early stage and hypothyroidism at the late stage whereas amiodarone only caused hypothyroidism. CONCLUSION: Both dronedarone and amiodarone can induce dyslipidemia and increase the levels of TC, LDL-c, and HDL-c, and these effects may be associated with thyroid dysfunction.

[Purification and characterization of adult optic nerve head astrocytes].

OBJECTIVE: To establish a method of purifying and characterizing adult astrocytes from optic nerve head (ONH). METHODS: Experimental study. The lamina cribrosa tissue from ONH of human eye was isolated under anatomic microscopy, and then 4 to 6 little explants were incubated in each culture plate containing culture medium DMEM/F12. After 8 to 10 weeks, the cells were removed by digesting cells with 0.25% trypsogen. Selective astrocyte culture medium is subsequently used. After two passages, astrocytes were identified by the observation of cell morphology and immunofluorescent staining of GFAP and NCAM. RESULTS: After 2 to 3 weeks of explants planting, cells showed an obvious migration procession by crawling in succession from the verge of the explants and rapidly splitting. Most cells displayed a flat star shape or polygon after digested with trypsogen. Several cells are long fusiformis. Almost all cells presented a flat star shape and simultaneously expressed GFAP and NCAM when the cells cultured with selective astrocyte culture medium. CONCLUSIONS: Cultured human ONH astrocytes can be obtained by precisely separating lamina cribrosa and placing the explants on the margin of culture medium, a method that promotes cell adherence. Using selective astrocyte culture medium is very effective and convenient in purifying primary astrocytes.

[Progress of research on corneal transient receptor potential channel superfamily].

Cornea senses thermal, mechanical and chemical stimuli via various nociceptors. In the early years, knowledge of the mechanism of corneal nociception was merely restricted to neurophysiology, which classifies nociceptors according to the clusters of nerve fibers which transmit identical stimulation signals. During the past decade, benefiting from the promptly developing molecular biology and technology, classification of the nociceptors has been modified into the level of cell sensors, among which, transient receptor potential (TRP) channel superfamily weighs the most. Researches have shown that there are a couple of TRP superfamily members expressed in cornea, which play major roles in the process of corneal pain, neurotrophic keratopathy, epithelial wound healing and immuno-inflammation. Some TRPs have even been recognized as targets of management of keratopathy.

[Morphological characteristics of cornea in patients with vernal keratoconjunctivitis by in vivo laser scanning confocal microscopy].

OBJECTIVE: To explore the morphological characteristics on cornea in patients with vernal keratoconjunctivitis (VKC) by the application of in vivo laser scanning confocal microscopy (LSCM). METHODS: The experimental design was retrospective observation case series (case control study). Twenty-six patients, each diagnosed as bilateral VKC, were enrolled in the study, among which 13 were tarsal form, 5 were bulbar form and the rest were mixed form. Nine patients had the clinical course less than one year, eight subjects longer than three years, and the rest between them. Another twenty-six healthy volunteers with matching age and gender were selected as normal control. All participants had their right eyes examined with the in vivo confocal microscopy (HRT II/RCM). Central cornea and superior peripheral cornea were chosen as the examination points. The images were recorded automatically and cellular density of each layer was analyzed by installed software. Software ImageJ was utilized to analyze the density, diameter, branch number and tortuosity of subbasal nerve fiber in VKC patients. Independent t test was performed to assess the differences on cellular density between VKC patients and normal control, as well as those between central and peripheral cornea in VKC patients. Fisher chi-square test was used to compare the infiltration rate of Langerhans cells in corneal epithelium between VKC patients and controls. ANOVA was applied to assess the differences in cellular density among three subtypes, as well as among different duration of VKC. Independent t-test and chi-square test were applied to analyze the parameters of subbasal nerve fiber. RESULTS: The morphological changes in cornea included the absence of superficial hyperreflective polygonal epithelial cells, infiltration of Langerhans cells in and(or) underneath corneal epithelium and activation of keratocytes in anterior stroma. Corneal epithelium conjunctivalization and stromal neovascularization could be identified in patients with corneal neovascular epithelium. Longitudinal or oblique dark striae could be found in the posterior stroma in patients with complicated keratoconus. The density of epithelial cells at central and peripheral cornea in healthy controls were (6033.1 ± 998.7) cells/mm(2) and (6098.4 ± 298.3) cells/mm(2), while that in VKC patients were (5972.2 ± 1148.2) cells/mm(2) and (6178.5 ± 318.9) cells/mm(2) respectively, the differences being no statistical significant between them (t = 1.191, 1.011; P = 0.238, 0.318). However, it's found in VKC patients that cellular density at peripheral cornea was significantly higher than that at central area (t = 2.249, P = 0.03). The density of anterior stromal cells at central and peripheral cornea in healthy controls was (1001.4 ± 125.3) cells/mm(2) and (924.6 ± 201.4) cells/mm(2), while that in VKC patients was (1184.5 ± 115.3) cells/mm(2) and (1101.4 ± 151.1) cells/mm(2), the difference bearing no statistical significance (t = 6.617, 3.439; P = 0.001). The density of posterior stromal cells in normal subjects and VKC patients was (537.7 ± 42.6) cells/mm(2) and (548.7 ± 79.8) cells/mm(2), that of endothelial cells was (2985.7 ± 401.2) cells/mm(2) and (3021.5 ± 383.3) cells/mm(2), respectively, neither difference had statistical significance (t = 0.174, 1.112; P = 0.864, 0.282). Langerhans cell infiltration could be identified in 61.5% (16 cases) VKC patients, which was significantly higher than normal control (2 cases, 7.7%) (χ(2) = 12.49, P = 0.001). Furthermore, much intense Langerhans cells infiltration was found in bulbar form and mix form than tarsal form. (t = 6.617, P = 0.001). The density and diameter of subbasal nerve fiber in VKC patients decreased significantly than those in normal subjects, whereas the tortuosity increased significantly. CONCLUSIONS: The morphological changes of cornea in VKC patients mainly involve corneal epithelium, subbasal nerve fiber and anterior stroma. In vivo LSCM is helpful in discriminating the subtypes of VKC.

[The effects of hyperosmotic stress on rabbit ocular surface and mucin 5AC expression].

OBJECTIVE: To investigate the effects of hyperosmotic stress on rabbit ocular surface and mucin 5AC (MUC5AC) expression. METHODS: Experimental study. Eighteen New Zealand white rabbits were randomly divided into three groups with equal number as hyperosmolar saline solution (HOSS, 500 mmol/L) group, normal saline (NS, 308 mmol/L) group and blank control group respectively. In HOSS and NS groups, the HOSS and NS eye drops were instilled on bilateral eyes six times every day for 14 days. On day 0, 7 and 14, Schirmer I test and tear break-up time (BUT) were measured and conjunctival impression cytology specimens were collected. On day 7 and 14, cornea and conjunctiva were harvested for Hematoxylin and Eosin (HE) staining, scanning and transmission electron microscopy observation and conjunctival TUNEL examination. On day 14, the conjunctiva were also harvested for immunology histological staining and western blot to evaluate the expression of MUC5AC. RESULTS: In HOSS group, the BUT on day 7 and 14 was (7.6 ± 2.5) and (7.0 ± 2.3) s respectively which was significantly shorter than the (10.3 ± 2.5) s on day 0(t = 5.800, 4.950; P < 0.01), and also significantly shorter than the BUT in NS and control groups (F = 8.030, P < 0.01). But the Schirmer I test value did not change obviously in and between all those three groups. The mean conjunctival goblet cell (GC) density in HOSS group on day 7 and 14 was (19.5 ± 16.6) and (32.3 ± 18.2) cells/mm(2) respectively which was also significantly lower than the (75.7 ± 43.4) cells/mm(2) on day 0 (t = 5.319, 2.970; P < 0.05). However the GC density did not change obviously in other two groups with time. After instillation of HOSS for 14 days, subepithelial inflammatory cell infiltration was showed on conjunctival tissue specimens and decreased epithelial layers and evident desquamation were found in the cornea specimens by the HE staining. Under the electron microscope, decreased microvilli and loosened intercellular junction in the superficial epithelium and increased autophagic vesicles in basal epithelium were observed in the cornea in HOSS group; and decreased microvilli and mucous granules were found in the conjunctiva in HOSS group. Obvious TUNEL positive staining was showed in the conjunctiva in the HOSS group. Also the MUC5AC immunology histological staining and western blot indicated decreased MUC5AC protein expression in HOSS group. CONCLUSION: Hyperosmotic stress destroyed the structure of ocular surface epithelium, induced the decrease of conjunctival goblet cell density and MUC5AC expression, and led to the decreased tear film stability.

[Cell sheet technology and its applications in corneal tissue engineering].

Cell sheet technology (CST) is based on the use of poly (N-isopropylacrylamide, PNIPAAm), which can be exhibit reversible hydration and dehydration of its polymer chains in response to temperature changes across the lower critical solution temperature(LCST)of 32°C. By reducing the incubation temperature to 20°C, all cultured cells are harvested as intact sheets along with their deposited extracellular matrix (ECM) due to the conversion of the grafted PIPAAm from hydrophobic to hydrophilic, as ECM remains present on the basal surface of the cell sheets, they can maintain cell viability and function as well as directly transplanted to tissue beds or even layered to create three-dimensional (3D) tissue-like structures without any scaffolds or sutures. The temperature-sensitive surfaces' preparation approaches, density, thickness, membrane additive ingredients and so on, all affect cell adhesion and proliferation. It can maintain cell viability and improve function by accelerating cell sheet detachment through changing the membrane compositions, density as well as types of graft substrate. With CST, cultured autologous/allogeneic corneal seed cells in vitro used as transplant sources can overcome the problems of immunorejection of transplanted tissues as well as donor organ shortages. So far, the cell sheet of limbal epithelium and autologous oral mucosal epithelium obtained by the CST have been successfully used in clinical graft for ocular surface reconstruction. Finally, There is an overview of preparations of temperature-responsive surfaces, impacts of various factors that influenced cultured cells in vitro and clinical applications or clinically relevant animal experimentations of CST in corneal tissue engineering.

[The ocular surface of severe alkali burns patients on confocal microscopy].

OBJECTIVE: To analyze the morphology on the ocular surface of severe alkali burns patients by in vivo laser scanning confocal microscopy. METHODS: This research was a retrospective observation case series. From February to November 2008 in Eye Ear Nose and Throat Hospital of Fudan University, 39 alkali burns patients who classified as III or IV according to Roper-Hall classification were enrolled in this study. They were divided into four groups according to the course of disease: A (less than 3 months), B (3 - 6 months), C (6 - 12 months) and D (over 12 months). In vivo laser scanning confocal microscopic examinations were performed on the injured cornea, the limbus and the bulbar conjunctiva and the images were recorded. The morphology of the injured cornea, the limbus and the bulbar conjunctiva was analyzed and the densities of the inflammatory cells and dendritic cells in the limbus were calculated. One-way analysis of variance was used to compare the means of the inflammatory cells and dendritic cells. Subsequently the data between two groups were analyzed by least significant difference. RESULTS: The corneal epitheliums of the patients in Group A manifested large irregular features with hyperreflective cytoplasm and hyporeflective nuclei, sometimes losing cell features. There were numerous small hyperreflective inflammatory cells in groups beneath the superficial epitheliums. Shallow corneal stroma was edema, and it was hard to discriminate the morphology of the stromal cells. Deep stromal cells were in the activated state. The images of the endothelial layer were unclear. In Group B and Group C, there were the same manifestation of the superficial epitheliums as the group A and it disappeared in Group D. The inflammatory cells beneath the superficial epitheliums reduced and some residual basal epitheliums and hyperreflective conjunctiva-like epitheliums were visible in Group B and Group C. In Group D, there were small oval tight-arranged cells with punctiform hyperreflective nuclei instead of normal corneal basal epitheliums. In Group B, it was still hard to discriminate the morphology of the shallow stroma cells. Deep stromal cells were still in the activated state. In Group C and Group D, corneal stroma was replaced by the fibrous tissues. The images of the endothelial layer were still unclear in the other groups. The Vogt palisades in the limbus of the severe alkali burns patients were destroyed in all groups. There were rich vascular nets in the limbus. The densities of the limbal inflammatory cells in four groups were (4023 +/- 343), (2975 +/- 246), (2652 +/- 375), (2679 +/- 299) cells/mm(2), respectively. Significant difference in inflammatory cell density was found among groups (F = 40.001, P = 0.000). The densities of the limbal dendritic cells in four groups were (106 +/- 19), (132 +/- 35), (141 +/- 26), (98 +/- 24) cells/mm(2), respectively. Significant difference in dendritic cell density was found among groups (F = 8.053, P = 0.000). When the injured area of the conjunctiva was limited, it was hard to discriminate the morphology of the conjunctival epitheliums in both Group A and Group B. Numerous inflammatory cells infiltrated in the conjunctival lamina propria and goblet cells were invisible. In Group C and Group D, the conjunctival epitheliums were almost normal. There were still some inflammatory cells and dendritic cells in the conjunctival lamina propria, and there were residual goblet cells visible in parts of the patients. When the injured area of the conjunctiva was large, the conjunctivas in four groups displayed hyperreflective stripe fibrous tissues instead of normal conjunctival epitheliums. CONCLUSIONS: The application of laser scanning confocal microscopy indicates that there is much difference on the cellular morphology of the ocular surface of severe alkali burns patients among diverse courses of the disease. The technique is a useful tool to the observation on the ocular surface of severe alkali burns patients.

[Advances in penetrating keratoplasty technique].

Rejection after corneal transplantation is currently the lead of the main reasons for corneal graft failure. Penetrating keratoplasty (PK) is still one of the gold standard surgeries for these patients. In recent years, improving the surgical techniques to reduce the incidence of corneal allograft rejection has been the way to more and more attention to the majority of ophthalmologists. To generalize and analyze the recent research of new technologies of femtosecond laser shaped penetrating keratoplasty of top-hat configuration (FS-TH-PKP), manual top-hat wound configuration for penetrating keratoplasty (M-TH-PKP), and half-top-hat penetrating keratoplasty (HTH-PKP) surgeries.

An in vivo confocal microscopy and impression cytology analysis of goblet cells in patients with chemical burns.

PURPOSE: To evaluate goblet cell density (GCD) on conjunctiva and cornea in patients with ocular chemical burns by in vivo laser scanning confocal microscopy (LSCM) and impression cytology (IC) and to explore the correlation between two methods. METHODS: Fifty-four patients (58 eyes) with chemical burn were enrolled in the study. LSCM was applied to identify the goblet cells on conjunctiva and cornea under in vivo conditions, and GCD was analyzed with the customized software. Impression cytology was then performed, and the biopsy specimens were stained to visualize goblet cells in vitro and to measure the density. Statistical software was used to analyze the correlation between GCD taken by two methods. RESULTS: Conjunctival goblet cells could be discriminated in 55 eyes and 57 eyes by in vivo LSCM and IC. They could be identified on the cornea in nine eyes and eight eyes by two methods. The positive rate of two methods had no significant difference. GCDs on conjunctiva measured by in vivo LSCM and IC were 136 +/- 79 cells/mm(2) and 121 +/- 66 cells/mm(2). Median GCDs on cornea detected by two methods were 30 cells/mm(2) and 23 cells/mm(2), respectively. A significant positive correlation was found between the GCDs on conjunctiva measured by these two methods as well as the GCDs on cornea. CONCLUSIONS: GCD decreased in patients with chemical burns. A positive correlation was found between GCD measured by in vivo LSCM and IC after chemical burns. In vivo LSCM was a promising device to study goblet cells in vivo under pathologic conditions.

[Clinical assessment of oral ganciclovir capsules on the treatment of herpes simplex keratitis].

OBJECTIVE: To evaluate the clinical values of oral ganciclovir on the treatment of herpes simplex keratitis (HSK). METHODS: A randomized, controlled, single-blind and prospective study was carried out from May in 2008 to June in 2009 at Department of Ophthalmology, Eye Ear Nose and Throat Hospital of Fudan University. 60 patients (60 eyes) with HSK, including stromal keratitis and corneal endotheliitis, were enrolled in the study and were randomly arranged into two groups in average. Oral ganciclovir was orally administered 1000 mg 3 times per day for 8 weeks, 0.15% ganciclovir ophthalmic gel, 4 times per day, and 0.1% fluorometholone eye drops, 3 times per day, in the test group, meanwhile, the control group was adopted the same ophthalmic gel and eye drops without the oral capsules. The symptoms and signs were evaluated before and after the therapy 1st week, 2nd week, 4th week, 6th week and 8th day respectively with the side effects observed. RESULTS: There was no significant difference between the control and test group in the mean scores of symptoms (control 10.70 ± 3.61, test 11.87 ± 3.47) and signs (control 13.83 ± 3.74, test 15.27 ± 3.83) respectively before the treatment (Z = -1.269 and -1.419; P > 0.05). After the administration, the total scores of symptoms and signs in the test group were 8.37 ± 4.31, 2.70 ± 2.65, 0.70 ± 1.44, 0.33 ± 0.92 and 0.17 ± 0.65 respectively at each follow-up time point, which were obviously lower than those in the control group, 13.63 ± 7.64, 10.53 ± 7.18, 7.83 ± 6.49, 5.37 ± 5.33 and 4.37 ± 5.11 respectively (Z = -2.801, -4.895, -5.260, -4.758, and -4.292; P < 0. 05). The efficacy rates in the test group were all 100.0% after the administration, but those in the control group were 50.0%, 73.3%, 86.7%, 93.3% and 96.6%. Furthermore, the cure rates in the test group were 0.0%, 36.7%, 76.7%, 90.0% and 93.3% respectively at each follow-up time point, which were significantly higher than those in the control group with 0.0%, 3.3%, 16.7%, 30.0% and 43.3% respectively (χ(2) = 20.00, 16.433, 22.571, 22.636 and 17.330; P < 0. 001). There was no obvious discomfortableness and adverse reaction observed in the test group. Unfortunately, 5 patients in the control group and 3 patients in the test group underwent the recurrence of HSK after the course of treatment, but there was no significant difference between the groups in the recurrence rate. CONCLUSIONS: Oral ganciclovir can effectively assist to relieve the symptoms and signs and shorten the pathogenesis of herpes simplex stromal keratitis and corneal endotheliitis. And short-term oral ganciclovir has confirmed good safety.

[Normal human bulbar conjunctiva on confocal microscopy in vivo].

OBJECTIVE: To analyze the morphology of human bulbar conjunctiva by in vivo laser scanning confocal microscopy. METHODS: This research was a cross-sectional study. From February to July 2008, 50 eyes of 50 healthy subjects were enrolled in this study. They had no history of ocular trauma, infection or contact lens wear and had no found after routine slit-lamp examinations. In vivo laser scanning confocal microscopic examinations were performed on the superior, inferior, nasal and temporal bulbar conjunctiva and the images were recorded. The morphology of bulbar conjunctiva was analyzed and the density of epithelial cells, dendritic cells and goblet cells were calculated. One-way analysis of variance (ANOVA) was used to compare the means of epithelial cell densities in different layers and goblet cell densities in different positions. Subsequently the datum between two groups were analyzed by least significant difference (LSD). RESULTS: Superficial epithelial cells of bulbar conjunctiva were characterized as large loose-arranged cells with a hyporeflective nucleus. The mean density is (1643 +/- 206) cells/mm(2). Intermediate epithelial cells were captured with features of oval small tight-arranged cells with a punctiform hyperreflective nucleus. The mean density is (4693 +/- 228) cells/mm(2). Basal epithelial cells appeared to be polygonal and regular-arranged within hyperreflective cell borders. The mean density is (4420 +/- 230) cells/mm(2). There was a significant difference among three kinds of conjunctival epithelium (F = 1160.312, P = 0.000). The presumed goblet cell was defined as a large hyperreflective oval-shaped cell with relatively homogeneous brightness, crowded in groups or mainly dispersed. The mean density is (432 +/- 72) cells/mm(2). The dendritic cell appeared to be hyperreflective corpuscular particles with dendritic processes scattered among conjunctival epithelial cells. The mean density is (22 +/- 25) cells/mm(2). The basement membrane, a prominent hyperreflective band, separated epithelial cells from subepithelial structure. Bulbar conjunctival substantia propria, beneath the basement membrane, was mainly composed of highly vascularized, loose connective tissues which were irregularly arranged fibers or a network of fibers, punctiform hyperreflective immune cells and sharp flows of blood vessels. CONCLUSION: In vivo laser scanning confocal microscopy is a useful tool in the analysis of the bulbar conjunctival morphology, which provided a fast and noninvasive method for the diagnosis of ocular surface diseases.

[The influence of glaucoma filtration surgery on anterior chamber associated immune deviation in mice].

OBJECTIVE: To study glaucoma infiltration surgery induced the anterior chamber associated immune deviation (ACAID) by with or without anterior chamber (AC) implantation of foreign epithelium removed corneal graft in mice. METHODS: In this study, fifty Wistar mice were randomly divided into five groups (ten in each group) and additional five SD and one Wistar mouse were used as the providers of epithelium removed corneal grafts to induce ACAID as follows: Group A: corneal grafts from Wistar mouse were implanted into the AC 1 week after the infiltration surgery. Group B: the spleen cells were injected into the nape of neck 1 week after the glaucoma infiltration surgery. In group C, D, and E, the corneal grafts from SD mice were implanted into the AC at 1 week, 4 week, and 8 week after the glaucoma infiltration surgery, respectively. To induce the delayed type hypersensitivity (DTH), spleen cells were injected into the right ear pinna 1 week after the neck injection in group B, 2 weeks after the AC implantation of corneal grafts in other groups. At the same time points as the induction of DTH, heart blood was collected to detect the concentration of IL-4 and IL-10. The spleens were removed to evaluate the expression of the GATA-3 mRNA by RT-PCR. The eyeballs were enucleated and used to evaluate the histopathological changes. RESULTS: (1) The DTH were found in group B, C, D, and E with corneal grafts of SD, but not in group A with that of Wistar. (2) The serum concentrations of IL-4 and IL-10 were statistically different in group C, D, and E (F = 49.124, 6.336; P < 0.01). The concentrations of IL-4 and IL-10 were significantly (P = 0.002) lower in group D than those in group C and E (3.759 +/- 0.250 vs 5.916 +/- 0.500 or 4.566 +/- 0.518, and 17.170 +/- 3.943 vs 44.447 +/- 17.167 or 35.643 +/- 21.233 microg/L, n = 10, respectively), which showed a biphase response fluctuation of ILs. (3) The GATA-3 mRNA was obviously up-regulated in group D and E. (4) The exudation and a few inflammatory cells in the AC were observed in group C, but not in group D and E. CONCLUSIONS: The eye with the glaucoma infiltration surgery results in a temporary incapable of inducing ACAID, which is gradually recovered following the diminish of intraocular inflammation and the increase of levels of IL4, IL-10 and GATA-3 mRNA.

[Proliferative ability and ultramicrostructure of basal limbal epithelial cells in different age group of normal subjects].

OBJECTIVE: To demonstrate the difference in proliferative ability and ultramicrostructure of basal limbal epithelial cells in different age group of normal subjects. METHODS: It is a case-control study.40 specimens of limbus from 40 eyes provided by the eye bank of Eye Ear Nose and Throat Hospital of Fudan University in 2007 - 2008 were enrolled in this study. Only one eye was enrolled from the same donner. Specimens were divided into 4 groups according to the donners' age: A (0 - 19 years), B (20 - 39 years), C (40 - 59 years) and D (60 - 79 years). Immunohistochemistry of proliferating cell nuclear antigen (PCNA) was performed on 10 specimens of each group. The staining status of limbal epithelium was observed and the staining positive rate of the limbal basal epithelial cells was graded. And transmission electric microscopy was performed on specimens of each group. The morphologic feature of the ultramicrostructure of limbal basal epithelial cells were observed. RESULTS: 50% of specimens in group A showed a strong PCNA staining of (++++), while a weak staining of (+) took a main part in all the other 3 groups (B 40%, C 60% and D 50%, respectively). Furthermore, positive nuclear staining of limbal superficial cells was observed in 3 specimens and positive cytoplasm staining was observed in 1 specimen, all in group A. Transmission electric microscopy showed that the stem cell-like cells in group A possessed 3 morphologic features: extremely small, stretching along the basal membrane, with densely packed heterochromatin. In contrast, the stem cell-like cells in the other 3 groups did not show great disparity in cell size with their neighbor epithelial cells, and they stretched vertically to the basal membrane, with dispersed euchromatin. CONCLUSION: Difference in proliferative ability and ultramicrostructure of basal limbal epithelial cells was found in different age group of normal subjects.