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Background: Corneal blindness resulting from various medical condition affects millions worldwide. The rapid developing field of tissue engineering offers the potential to develop a tissue-engineered cornea that adheres very closely to the native cornea for transplantation. The design of a scaffold with mechanical properties and transparency similar to that of natural cornea is vital for the regeneration of corneal tissues. Hence, there is a need to investigate this relatively inexpensive but an improved scaffold to assist in human corneal stem cells delivery. Aim and Study Design: The present study aimed at to prepare and investigate the properties of poly vinyl alcohol (PVA)/chitosan blended scaffold by further cross-linking with 1-Ethyl-3-(3-dimethyl amino propyl)-carbodiimide (EDC), 2 N-Hydroxysuccinimide (NHS) as potential in vitro carrier for human limbal epithelial cells delivery. Results: After the viscosity measurement, the PVA/Chitosan scaffold was observed by Fourier transform infrared spectroscopy (FT-IR). The water absorbency of PVA/Chitosan was increased 361% by swelling. Compression testing demonstrated that by increasing the amount of chitosan, the strength of the scaffold could be increased to 16 × 10−1 Mega Pascal Pressure Unit (MPa). Our degradation results revealed by mass loss shows that scaffold degraded gradually implies slow degradation but shown enhanced the biomechanical properties. In vitro 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay showed good cell proliferation and growth in the scaffold. Besides the above, the present study aimed at exploring the effects if any observed with PVA/chitosan and other cross linkers on cell morphology and phenotype using H&E staining. Our MTT assay results and the cells observed on these membranes confirmed that the safer and improved method of preparation of membrane could increase the cells adhesion and growth on the substrata. Conclusion: Hence, we strongly believe the use of this improved PVA/chitosan polymer scaffold has potential to cut down the disadvantages of human amniotic membrane (HAM) for corneal epithelium in ocular surface surgery in future after successful experimentation with clinical trials.
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Objective To investigate the value of corneal laser confocal microscope in the diagnosis of corneal subepithelial plexus,corneal cell density and morphological changes of patients with diabetic retinopathy (DR).Methods Together 94 cases of confirmed DR (114 eyes),including 41 cases of non-proliferative diabetic retinopathy (NPDR group,52 eyes) and 53 cases of proliferative diabetic retinopathy (PDR group,62 eyes) were selected from January 2016 to April 2017,and meanwhile,40 diabetic patients (40 eyes) with no fundus abnormality were grouped as control group.Corneal laser confocal microscopy was used to compare the corneal subepithelial plexus,corneal cell density and morphological changes in the three groups,Results The cell densities of the basal layer,the superficial stromal layer,the medium stromal layer and the deep stromal layer of the cornea in NPDR and PDR were significantly lower than those of the control group (all P ＜ 0.05),and the PDR group was significantly lower than the NPDR group (all P ＜ 0.05).The corneal endothelial cell density,hexagonal cell ratio,nerve fiber density,and nerve fiber length in the NPDR and PDR group were significantly lower than those in the control group (all P ＜ 0.05);and the variability of endothelial cell and nerve branch density in NPDR and PDR patients were significantly higher than those in the control group (all P ＜ 0.05).The corneal endothelial cell density,hexagonal cell ratio,nerve fiber density,and nerve fiber length in the PDR group was (1962.0-± 117.3) · mm-2,46.1％ ± 5.5％,(15.4 ± 3.3) · mm-2,(6.2 ± 2.7) mm · mm-2,respectively,which were significantly lower than those in the NPDR group [(2381.4 ± 144.0) · mm-2,58.2％ ±7.0％,(20.6 ±3.8) ·mm-2,(8.6 ± 2.4)mm · mm-2,respectively] (all P ＜ 0.05),but the variability of endothelial cell and nerve branch density in PDR group were significantly higher than those in the NPDR group (all P ＜ 0.05).Conclusion Corneal confocal microscopy can effectively observe the density and morphological changes in corneal subepithelial plexus and corneal cell in DR patients so as to provide guidance for clinical diagnosis and treatment.
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Autophagy and apoptosis function as important early cellular defense mechanisms in infections and other diseases. The outcome of an infection is determined by a complex interplay between the pathogenic microorganism and these intracellular pathways. To better understand the cytopathogenicity of Herpes simplex virus types 1 and 2 (HSV-1 and - 2), we studied the effect of these viruses on the autophagic and apoptotic processes in the SIRC corneal cell line. Infection with the KOS strain of HSV-1 and a wild-type strain of HSV-2 enhanced autophagosome formation, triggered cytoplasmic acidification, increased LC3B lipidation and elevated the ratio of apoptotic cells. The autophagy inhibitor bafilomycin A1 triggered a significant increase in the apoptotic responses of HSV-1- and HSV-2-infected cells. Thus, both HSV types affect autophagy and apoptosis in a coordinated fashion, and autophagy plays cytoprotective role in HSV-infected cells via antagonizing apoptosis. Together these data implicate autophagy in the pathogenic mechanism of herpetic keratitis.
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Aims: To understand the mechanisms of Early Growth Response Protein 1 (Egr-1) induction upon HSV-1 lytic infection and its roles in regulating viral gene expression and replication. Study Design: Rabbit corneal cell line SIRC and other cell lines were infected by HSV-1 to investigate the Egr-1 induction and its occupancy on the viral genome in different conditions. UV-inactivated HSV-1 and a recombinant virus over-expressing Egr-1 were generated to evaluate the regulatory effects on viral gene expression and replication during the infection. Methodology: Egr-1 induction triggered by viral infection was determined by Western Blot analyses and immune-fluorescent microscopy. Real-time RT-PCR and a novel Cignal™ Reporter Assay were used for quantitative measurement of Egr-1 expression. Chromatin Immuno-precipitation (ChIP) was performed to address the Egr-1 occupancy to the viral regulatory sequences and the influence on viral replication was assessed by plaque assays. Results: Our results indicated that Egr-1 expression requires viral gene expression since the UV-inactivated HSV-1 failed to produce Egr-1 protein. Blockade of viral replication did not block the Egr-1 protein synthesis, supporting the hypothesis that HSV-1 replication was not essential for Egr-1 production. Chromatin immune-precipitation (ChIP) and RT-PCR assays demonstrated that induced Egr-1 was able to interact with key regulatory elements near HSV-1 immediate-early (IE) genes and promote viral gene expression. Recombinant virus overexpressing Egr-1 revealed that Egr-1 enhanced the viral replication and the release of infectious virus. Conclusion: Together these results concluded that HSV-1 triggers the expression of an important host transcription factor Egr-1 via a unique mechanism and benefit the viral gene expression and replication.
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Objective To analyze the stability of chromosome variant ratio of three available transformed corneal cell lines. Methods Chromosome specimens of transformed cells including human corneal epithelial cells(HCE),bovine corneal endothelial cells(BCE) and rabbit corneal epithelial cells(RCE) were prepared by a direct method using regular Giemsa staining. Chromosomes of cells in metaphase were counted under the microscope. Then, the variant ratio of chromosomes and their nuclear types were analyzed. Results The chromosome numbers were 56 to 65, 27 to 34 and 74 to 88 for HCE, BCE and RCE, respectively. Chromosome numbers in the three commonly used and transformed corneal cell lines were changed in comparison to their parent tissues. Conclusion Genotyping study may provide important information for using HCE、BCE、RCE in functional studies.
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In order to help define the possible role of adhesion molecules in corneal inflammation, we investigated the distribution of adhesion molecules in normal and Aspergillus fumigatus keratitis-induced corneas of rabbits in process if time. Each 4 corneas were resected at 3, 12, 24, and 72 hours after intracorneal injection with A. fumigatus. Normal corneas (4 eyes) were used as a control. Monoclonal antibodies to beta 1 subunit of VLAs, alpha 1 subunit of VLA-1, LFA-1, ICAM-1,and ELAM-1 were used for immunohistochemical staining of 20 corneas.The results were as follows: In normal cornea, beta 1 subunit of VLAs was expressed on all parts of the cornea, and ICAM-1 was expressed on corneal stroma and endothelium. Vascular endothelium showed expression of beta 1 subunit of VLAs and ICAM-1 after 12 hours of intracorneal injection, and alpha1 subunit of VLA-1 and ELAM-1 at 72 hours after injection. In inflamed cornea, beta 1 subunit of VLAs was expressed strongly at 72 hours after injection. Alpha1 subunit of VLA-1 was detected on corneal stroma after 12 hours of injection, and on corneal endothelium at 72hours after injection. Expression of beta 2 subunit of LFA-1 was weak on corneal stroma after 3 hours injection, and on corneal endothelium at 72 hours after injection. ICAM-1 expression was detected weakly on corneal epithelium, and increased on corneal stroma and endothelium at 72 hours after injection. ELAM-1 was expressed weakly on corneal stroma after 3 hours of injection, and on corneal endothelium at 72 hours after injection.In this study, it was found that the invasion of A. fumigatusinto the cornea causes localized inflammatory reaction that results in activation of corneal cells (keratocytes, corneal endothelial cells and epithelial cells), and subsequent expression of adhesion molecules in the cornea. Expression of adhesion molecules facilitates the inflammatory cells to be migrated into the cornea with inflammmation.