高脂血症兔球结膜微循环的改变

本实验用胆固醇粉喂家兔,造成实验性高脂血症,活体观察球结膜微循环的形态、流态及血管周围状态等１５项指标,并用球结膜微循环综合定量评价方法计算了综合积分值。处死后取球结膜进行组织学检查。结果表明：高脂血症家兔球结膜微循环有明显改变,主要为微血管形态异常、血流减慢、红细胞聚集、白微栓、静脉壁上有白色斑块等改变。球结膜微循环综合积分值明显增加,高于实验前。球结膜组织学检查发现上皮层下有泡沫细胞、血管壁增厚、有空泡、静脉内有血栓。虹膜睫状体内除有多数泡沫细胞外,还有胆固醇的菱形结晶。上述改变表明,高脂血症兔球结膜微循环有明显改变。血液有高凝、高聚倾向。黑茶中的茶色素有抗凝、抑制血小板聚集、促进纤溶等作用,口服黑茶可改善高脂血症兔球结膜微循环障碍。     

In vitro analyses of the anti-fibrotic effect of SPARC silencing in human Tenon's fibroblasts: comparisons with mitomycin C

Failure of glaucoma filtration surgery (GFS) is commonly attributed to scarring at the surgical site. The human Tenon's fibroblasts (HTFs) are considered the major cell type contributing to the fibrotic response. We previously showed that SPARC (secreted protein, acidic, rich in cysteine) knockout mice had improved surgical success in a murine model of GFS. To understand the mechanisms of SPARC deficiency in delaying subconjunctival fibrosis, we used the gene silencing approach to reduce SPARC expression in HTFs and examined parameters important for wound repair and fibrosis. Mitomycin C-treated HTFs were used for comparison. We demonstrate that SPARC-silenced HTFs showed normal proliferation and negligible cellular necrosis but were impaired in motility and collagen gel contraction. The expression of pro-fibrotic genes including collagen I, MMP-2, MMP-9, MMP-14, IL-8, MCP-1 and TGF-β(2) were also reduced. Importantly, TGF-β(2) failed to induce significant collagen I and fibronectin expressions in the SPARC-silenced HTFs. Together, these data demonstrate that SPARC knockdown in HTFs modulates fibroblast functions important for wound fibrosis and is therefore a promising strategy in the development of anti-scarring therapeutics.     

The differences in the eyelids microstructure and the conjunctiva‐associated lymphoid tissue between selected ornamental and wild birds as a result of adaptation to their habitat

The aim of the study was to describe the morphology of the upper, lower and third eyelid and characterize the organized lymphoid follicles and diffuse lymphocytes from ornamental and wild birds. The goal of these examinations was also to identify avian conjunctiva‐associated lymphoid tissue (CALT) and lymphoid tissue that contained specialized high endothelial venules. The upper, lower and third eyelid from 30 species of ornamental and wild birds representing 21 families were examined under light microscopy and using scanning electron microscopy. The third eyelid in all of the examined birds was composed of a free margin, which was divided into two parts. The largest tarsal plate of the third eyelid was observed in the greater rhea (Rheimorphae), the white‐tailed eagle and steppe eagle (Accipitrimorphae) and was approximately 13–15 mm wide and 9–11 mm long, respectively. In all of the examined birds, the CALT was associated with a rich network of small vessels. In addition, the presence of characteristic high endothelial venules and roundish bright endothelial cells was confirmed in the upper and lower eyelids or only in the lower eyelid (Phoenicopterimorphae, Procellariimorphae and Strigimorphae).     

Functional morphology of mucosal goblet cells based on spatial separation of orifice openings to the surface – Application to the rabbit bulbar conjunctiva

The purpose of the study was to assess spatial separation of goblet cell orifices observed at the surface of the rabbit bulbar conjunctiva by scanning electron microscopy (SEM) specimens of the bulbar conjunctiva from 8 healthy pigmented rabbits were obtained using a special preparation technique by which the tissue was carefully stretched out during glutaraldehyde fixation. On high magnification prints of SEM images of the conjunctival surface, the locations of goblet cell openings (orifices) to the apical surface were marked and the centre-to-centre spacing between all such orifices measured. Across the regions of interest (ROI), with averaged dimensions of 322 μm × 230 μm (adjusted for tissue shrinkage), the averaged value for the distances between all orifices was 196 μm (range 141–241 μm), with the calculated density of orifices being 412 mm⁻². A sequential order-based analysis of the spatial separation between orifices indicated a predictable value of 6 μm, a separation that showed a nearly linear inter-dependence over distances of at least 200 μm. The openings of goblet cells to the surface of unstimulated bulbar conjunctiva have a organized spatial distribution that is consistent with there being an organized control of goblet cell secretion.     

Giemsa-based cytological assessment of area,shape and nucleus:cytoplasm ratio of goblet cells of rabbit bulbar conjunctiva

Goblet cells were visualized in impression cytology specimens from bulbar conjunctiva of the rabbit eye using Giemsa staining. Highly magnified images were used to generate outlines of the goblet cells and their characteristic eccentric nuclei. Using sets of 10 cells from 15 cytology specimens, I found that the longest dimension of the goblet cells averaged 16.7 ± 2.3 μm, the shortest dimension averaged 14.4 ± 1.8 μm and the nucleus averaged 6.3 ± 0.8 μm. The goblet cells were ellipsoid in shape and the longest:shortest cell dimension ratio averaged 1.169 ± 0.091. The goblet cell areas ranged from 108 to 338 μm² (average 193 ± 50 μm²). The area could be predicted reliably from the longest and shortest dimensions (r² = 0.903). The areas of goblet cell nuclei were 15–58 μm² (average 33 ± μm²) and the nucleus:cytoplasm area fraction was predictably greater in smaller goblet cells and less in the larger goblet cells (Spearman correlation = 0.817). The nuclei were estimated to occupy an average of 9.5% of the cell volume. The differences in size, shape and nucleus:cytoplasm ratio may reflect differences in goblet cell maturation.     

Efficacy of a training programme designed to teach cervical smear screeners to identify ocular surface squamous neoplasia using conjunctival impression cytology

Efficacy of a training programme designed to teach cervical smear screeners to identify ocular surface squamous neoplasia using conjunctival impression cytology
Ocular surface squamous neoplasia (OSSN) is a recently proposed term introduced to encompass both intraepithelial neoplasia and invasive squamous cell carcinoma of the conjunctiva and cornea. A teaching programme incorporating a manual, slide sets, and an evaluation test was developed. The aim was to teach experienced cervical smear screeners to evaluate ocular surface specimens collected by conjunctival impression cytology, with a minimum of individual tuition. The use of the manual was well accepted and half of the original six candidates were able to master the new skill adequately within 8 h. It was considered that the differences between the cytology of OSSN and the equivalent lesions of the uterine cervix are sufficient to prevent some experienced screeners acquiring these skills rapidly.     

Corneal epithelium as a platform for secretion of transgene products after transfection with liposomal gene eyedrops

BACKGROUND: The first objective of the study was to evaluate the transfection of corneal epithelium with non-viral vectors to secrete transgene products into the tear fluid and aqueous humor. The second goal was to evaluate the differentiated corneal epithelial cell culture for transfection studies. METHODS: The human corneal epithelial (HCE) cell line was cultured to different stages of differentiation and transfected with complexes of pCMV-SEAP2 with DOTAP/DOPE, DOTAP/DOPE/protamine sulfate (PS) and polyethylenimine (PEI). The complexes of DOTAP/DOPE with plasmid (CMV-SEAP2 or pCMV-Luc4) were subsequently applied topically to the rabbit eyes. Secreted alkaline phosphatase (SEAP) was analyzed using chemiluminescent assay. Luciferase (Luc) was detected at the mRNA level in cornea and conjunctiva using a qRT-PCR. RESULTS: The transfection levels decreased with differentiation of HCE cells. PEI was effective in transfecting both the dividing and partly differentiated cells, but ineffective in differentiated cells. DOTAP/DOPE showed high activity in differentiated cell cultures, while added PS did not improve transfection. Significant SEAP expression was observed for three days after in vivo transfection in the tear fluid and aqueous humor. The luciferase mRNA was found both in the cornea and conjunctiva. The rates of SEAP secretion from both the basolateral side of differentiated HCE cells and cornea in vivo were within the same range. CONCLUSIONS: Corneal epithelium can be transfected topically to secrete gene products to the tear fluid and aqueous humor. The differentiated HCE model is a useful tool in the evaluation of non-viral carriers for corneal transfection.     

Successful Reconstruction of Damaged Ocular Outer Surface in Humans Using Limbal and Conjuctival Stem Cell Culture Methods

When the ocular outer surface is badly damaged, subsequent corneal transplantation fails due to the absence of basal cells that are needed to support the graft. With the realization that the limbus and the conjunctiva have adult stem cells that can be cultured, it has been possible for us to explant culture these on de-epithelized human amniotic membrane, and to graft the resulting viable and transparent epithelium to 125 needy human patients with success. Ultrastructural, histological, biochemical and immunological assays establish the identity of the cells and the tissue formed.     

Clinical and light microscopic studies of the conjunctival tissues of dogs with bilateral keratoconjunctivitis sicca before and after treatment with topical 2% cyclosporine

Abstract

We determined the concentrations of goblet and immune cells in conjunctival imprints and tissues of canines with keratoconjunctivitis sicca (KCS) before and after cyclosporine A (CsA) treatment. Twelve dogs with bilateral KCS were assigned to three groups: untreated, treatment group 1, and treatment group 2. The treatment groups were treated topically with 2% ophthalmic CsA solution for 45 days; CsA treatment group 2 was followed for an additional 30 days after discontinuation of the drug. Schirmer tear test (STT) scores were recorded prior to CsA treatment and on alternate days throughout the experiment. CsA treatment improved the STT scores, restored conjunctival histology, increased goblet and epithelial cell numbers, and decreased numbers of inflammatory cells. Although the STT scores regressed slightly at day 30 after discontinuing the treatment, the scores were higher than the baseline values. Topical CsA treatment resolved clinical signs of KCS, improved STT scores and restored normal conjunctival histology.