In situ localization of cytoskeletal elements in the human trabecular meshwork and cornea

The authors compared cytoskeletal elements of the in situ human trabecular-meshwork cell with in situ human corneal cells using indirect immunofluorescence staining for tubulin and intermediate filaments (vimentin, cytokeratin, and desmin) and NBD-phallacidin staining for f-actin using both fixed frozen and unfixed frozen sections from postmortem eyes. Both f-actin and tubulin were found throughout the cell body of trabecular-meshwork cells, keratocytes, corneal endothelium, and corneal epithelium. The f-actin staining pattern was concentrated at the cell periphery of these four cell types. Vimentin stain was intensely localized in focal areas of the trabecular-meshwork cell, keratocytes, and throughout the corneal endothelium. A general anticytokeratin antibody was intensely localized in corneal epithelium and endothelium. However, PKK-1 anticytokeratin antibody was seen only in superficial layers of corneal epithelium and not in corneal endothelium. The 4.62 anticytokeratin antibody was not observed in either corneal epithelium or endothelium. None of these three cytokeratin antibodies were seen in trabecular-meshwork cells or keratocytes. Desmin stain was not noted in any of these cell types. In general, cytoskeletal staining of unfixed frozen sections showed a similar staining pattern for f-actin and tubulin but a more uniform and intense staining pattern for vimentin and cytokeratin compared with fixed frozen material. The authors conclude that these cytoskeletal stains can differentiate human trabecular-meshwork cells from cells of the cornea in situ.     

Growth and characterization of rabbit corneal cells in vitro

Primary organ cultures of rabbit corneal epithelium, stroma, and endothelium were established by microdissection. Secondary cultures of epithelial cells, keratocytes, and endothelial cells were established by serial passage. The doubling time for epithelial cells and keratocytes was 18 h, and endothelial cells doubled their number in 5 days. Ultrastructural studies demonstrated characteristic morphological, nuclear, and cytoplasmic features of corneal epithelial cells, keratocytes, and endothelial cells and confirmed the identity of the cell lines. The purity of the three distinct cell types was ascertained by indirect immunofluorescence techniques, using antibodies against keratin, which identified epithelial cells, and fibronectin, which identified keratocytes and endothelial cells. The indirect immunofluorescence technique represents a simple method to screen an aliquot of a cell suspension and determine the purity of corneal cells grown in vitro.     

The effect of long-term contact lens wear on the cells of the cornea.

PURPOSE: To review the effects of long-term contact lens wear on the cells of the cornea. METHODS: Published investigations into the effects of long-term contact lens wear on the corneal epithelium, keratocytes, and endothelium were reviewed. RESULTS: Results from multiple studies indicate that long-term daily wear of contact lenses causes endothelial polymegethism but has no effect on corneal epithelium (thickness, surface cell size); keratocytes (density, stromal reflectivity); or endothelial cell density, permeability, or pump rate. Extended wear also causes no endothelial changes other than polymegethism, but it affects the epithelium (decreased thickness, increased surface cell size) and keratocytes (possible decreased density). CONCLUSIONS: Investigators have not found detrimental effects on the cells of the cornea from the long-term use of daily wear contact lenses. Although contact lenses cause endothelial polymegethism, no functional deficits ensue. Extended wear lenses may cause changes in all three cell types, but it is not known if these effects are detrimental nor if they occur with newer lenses of higher oxygen transmissibility. Patients can be reassured that modern contact lenses can be worn for many years in daily wear fashion (and possibly in extended wear with lenses of very high oxygen transmissibility) without damaging the cells of the cornea.     

Dynamics of donor cell persistence and recipient cell replacement in orthotopic corneal allografts in mice.

PURPOSE. To determine the extent to which donor cells persist and recipient cells repopulate each of the three cell layers of orthotopic corneal grafts in mice. METHODS. BALB/c, C57BL/6, and enhanced green fluorescence protein (EGFP) transgenic mice (B6 background) were used as donors and recipients for orthotopic syngeneic and allogeneic corneal grafts. Graft-bearing eyes were harvested at 5, 10, 15, 28, and 56 days, stained with propidium iodide, and observed (layer by layer) by confocal microscopy. Bone marrow-derived cells in the grafts were assessed immunohistochemically. RESULTS. Donor epithelium was totally replaced by recipient epithelial cells within 15 days in both syngeneic and allogeneic grafts, whereas donor stromal keratocytes and endothelial cells were retained virtually intact in syngeneic grafts and in accepted allografts. In rejected allografts, neither donor-derived keratocytes nor endothelial cells were detected, and, instead, recipient-derived stromal fibroblasts, neovessels, and infiltrating leukocytes were heavily represented. The posterior surface of rejected grafts was devoid of corneal endothelium and was covered incompletely with bone marrow-derived cells of recipient origin. CONCLUSIONS. Whereas in mice graft-derived epithelium is largely irrelevant to corneal allograft outcome, persistence of donor-derived endothelium and keratocytes correlates perfectly with graft acceptance. Recipient endothelium is incapable of covering the posterior surface of accepted or rejected corneal grafts, whereas bone marrow-derived cells of recipient origin come to occupy this site in rejected grafts.     

Pharmacokinetic of topical cyclosporin A in the rabbit eye

3H labelled cyclosporin A (CS-A) eye drops (2% in castor oil) were applied in rabbits. When a single drop of 10 microliters was given CS-A reached a maximal concentration of 900-1400 ng/ml in the cornea 6 hr after application and then slowly decreased. A substantial level of 600-900 ng/ml was still present after 48 hr. When corneas were analysed for their cellular compartments, 67% of CS-A was found in the epithelium, 25% in the stroma (probably in keratocytes), and 8% in the endothelium. In corneas denervated by circular keratotomy, slightly reduced concentrations were found after surgery, the reduction was not therapeutically significant. Using the HPLC method a significant corneal metabolism of CS-A could be excluded. Scleral concentrations of CS-A as high as 180 ng/ml were found. Concentrations of CS-A in aqueous humor, lens, vitreous body and uvea/retina were below the therapeutic range.     

Herpes simplex virus type 1 persistence and latency in cultured rabbit corneal epithelial cells, keratocytes, and endothelial cells.

Cell cultures of rabbit corneal epithelium, keratocytes, and endothelium were used to determine the lytic cycle of herpes simplex virus type 1. Viral growth was fastest in epithelial cells. A novel HSV-1 in-vitro latency system was established in the three distinct cell types. Cell cultures were inoculated at low multiplicities of infection with HSV-1. Temperature manipulation alone was used to induce and reactivate latent HSV-1 infections. The presence of cellular stress proteins was demonstrated at supraoptimal temperatures. All cell types were capable of maintaining latent viral infections under these conditions. Viral persistence was present in 20% of epithelial cell cultures at supraoptimal temperatures, but not in keratocyte cultures or endothelial cell cultures.     

Toxicity of topical anesthetic agents to human keratocytes in vivo.

PURPOSE: To test the potential toxicity on human keratocytes of topical anesthetic agents used after photorefractive keratectomy (PRK) to reduce or eliminate pain. SETTING: Department of Ophthalmology, Doheny Eye Institute, University of Southern California, Los Angeles, California, USA. METHODS: Cultured human keratocytes were incubated with commercially available tetracaine and proparacaine at reduced concentrations of 0.001%, 0.01%, 0.1%, and 0.25%. Evaluations were performed by phase-contrast microscopy and tetrazolium salt colorimetric assay every 2 hours for 12 hours after adding 1 of the anesthetic agents to the media. RESULTS: After time of incubation and concentration were adjusted, both drugs reduced overall cell viability; however, tetracaine produced a larger decrease in cell viability than proparacaine (P = .008). For both drugs, significant differences were found among concentrations for and across time (P < .001 and P = .004, respectively). CONCLUSION: Both tetracaine and proparacaine had toxic effects on stromal keratocytes related not only to drug concentrations but also to time exposure. These findings underscore the widespread concern that anesthetic drugs may affect corneal stromal wound healing after PRK.     

Localization of lectin binding sites in human, cat, and rabbit corneas

Paraffin sections of human, cat, and rabbit corneas were stained with nine lectins, using an avidin-biotin-complex procedure to study glycoconjugates of the epithelium, keratocytes, and stromal matrix. Wheat germ agglutinin (WGA) stained plasma membranes of all epithelial cell layers of cat and human and superficial and wing cells of rabbit. Plasma membranes of superficial and wing cells of cat epithelium also stained with peanut agglutinin (PNA) and Ricinus communis agglutinin I (RCA-I). Human and cat keratocytes stained with WGA and RCA-I. Stromal matrices of all three species were stained with concanavalin A and lentil agglutinin. In neuraminidase-treated sections, the entire epithelium and keratocytes of all three species stained with PNA. Corneal sections from the three species did not stain with Bandeiraea simplicifolia I, Bandeiraea simplicifolia II, Ulex europeus I, and Soybean agglutinin. These data suggest the presence of oligosaccharides with: N-acetylglucosamine/sialic acid residues in cell membranes of corneal epithelium of all species studied and in the keratocytes of human and cat; terminal beta-galactose residues in cat and human keratocytes, beta-galactose-galactosamine chains in cat epithelial cell membranes; and sialic acid-beta-galactose-galactosamine chains in epithelial cell membranes and keratocytes of all three species.     

Serum-free spheroid culture of mouse corneal keratocytes

PURPOSE: To develop a serum-free mass culture system for mouse keratocytes. METHODS: Corneas of C57BL6/J mice were enzyme digested after the epithelium and endothelium were removed. Stromal cells were cultured in serum-free DMEM/F12 (1:1) containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and B27 supplement. Primary spheres were dissociated by trypsin and subcultured as suspended secondary spheres. Cells from postnatal day (P)6 to P10 spheres were subcultured onto plastic dishes or type I collagen gels for phenotype analysis. The expression of the keratocyte markers keratocan, aldehyde dehydrogenase (Aldh), and CD34, were analyzed by RT-PCR, and vimentin and alpha-smooth muscle actin (alpha-SMA) were examined by immunocytochemistry. RESULTS: Primary keratocytes formed spheres, which were cultured for over 12 passages. Suspended sphere cells expressed vimentin, keratocan, CD34, and lumican, but were negative for cytokeratin K12 (K12) and Pax6. Sphere cells subcultured on plastic exhibited a dendritic morphology characteristic of keratocytes, and maintained keratocan, Aldh, and CD34 expression in serum-free medium. Sphere cells subcultured with 10% serum became fibroblastic, and expressed alpha-SMA when stimulated by transforming growth factor (TGF)-beta. alpha-SMA-positive cells demonstrated contractile properties on collagen gels, compatible with the myofibroblast phenotype. CONCLUSIONS: The phenotype of mouse keratocytes can be maintained in vitro for more than 12 passages by the serum-free sphere culturing technique.     

A placebo-controlled blind trial of cyclosporin-A in prevention of corneal graft rejection in rabbits.

Cyclosporin-A (CyA) administered to rabbits intramuscularly in a dose of 25 mg/kg/day for 14 days following interlamellar corneal grafting had a significant effect in preventing rejection of the corneal graft (p less than 0.05), and the benefit was maintained. Rejection was attended by an initial mild inflammatory reaction and followed by a protracted intense response involving a variety of cell types, with subsequent loss of epithelium. endothelium and keratocytes, and the development of areas of stromal necrosis. CyA suppressed this rejection response.     

灯盏花素通过调节Bcl-2和Bax表达拮抗高糖诱导的RPE细胞损伤

目的:探讨灯盏花素对高糖环境下人视网膜色素上皮(RPE)细胞氧化应激的影响。方法:常规培养RPE细胞,分为对照组(葡萄糖浓度为5.5mmol/L)、高糖组(葡萄糖浓度为30mmol/L)、灯盏花素低浓度组(30mmol/L葡萄糖+1μmol/L灯盏花素)和灯盏花素高浓度组(30mmol/L葡萄糖+10μmol/L灯盏花素)。细胞划痕实验观察RPE细胞的迁移性,CCK-8法检测细胞活性,流式细胞术检测细胞内活性氧(ROS)水平变化,Hoechst染色法观察凋亡细胞比例,Western blot分析各组Bcl-2和Bax的变化。结果:细胞划痕实验结果显示,灯盏花素低浓度组和灯盏花素高浓度组RPE细胞形态较高糖组改善,细胞迁移性较高糖组增加;CCK-8结果显示,用1、10μmol/L浓度的灯盏花素处理后,RPE细胞存活率分别提高到61.06%±5.59%和79.81%±7.04%,与高糖组(40.63%±4.72%)比较有差异(P<0.05);2’,7’-二氯二氢荧光素二乙酸酯(H2DCFDA)荧光探针染色结果显示,灯盏花素抑制了RPE细胞内ROS水平;Hoechst染色法显示,1、10μmol/L灯盏花素处理后发生凋亡的RPE细胞数量逐渐减少;Western blot结果显示,灯盏花素增强了Bcl-2蛋白的表达,降低了Bax蛋白的表达。结论:灯盏花素可以抑制高糖诱导的RPE细胞氧化损伤和凋亡的发生,为糖尿病视网膜病变的治疗靶点研究提供了理论支持。     

Immunohistochemical Expression of Fas Ligand (FasL) and Neprilysin (Neutral endopeptidase/CD10) in Keratoconus

Recent evidence demonstrated a correlation between apoptosis and neprilysin expression. The aim of this study was to investigate the immunohistochemical expression of Fas ligand (FasL) and neprilysin in keratoconic corneas in comparison to normal cadaver corneas to evaluate if such molecules play a role in the pathogenesis of keratoconus. We studied the expression of FasL and neprilysin in corneal specimens removed during penetrating keratoplasty in 15 cases with keratoconus and compared them with 5 normal cadaver corneas. In keratoconus, FasL was expressed in epithelium, endothelium and sub-Bowman’s stroma only, while neprilysin was expressed in epithelium, endothelium and all stromal layers. All normal corneas showed weak expression of both markers in basal epithelial layer only. In keratoconus, corneal epithelium with higher expression of FasL may evoke apoptosis in keratocytes, while neprilysin could prevent possible rescue of keratocytes from apoptosis.     

Mesothelial proteins are expressed in the human cornea

The goal of our study was to determine whether proteins typical of the human mesothelial cell phenotype, such as mesothelin, HBME-1 (Hector Battifora mesothelial cell-1) protein and calbindin 2, are expressed in the human cornea, especially in endothelial cells.Cryosections and endothelial and epithelial imprints of sixteen human cadaverous corneoscleral discs were used. The presence of proteins was examined using immunohistochemistry and Western blotting, while mRNA levels were determined by qRT-PCR.A strong signal for mesothelin was present in the corneal epithelium, while less intense staining was visible in the endothelium. Similarly, higher and lower mRNA levels were detected using qRT-PCR in the corneal epithelium and endothelium, respectively. HBME-1 antibody strongly stained the corneal endothelium and stromal keratocytes. Marked positivity was present in the corneal stromal extracellular matrix, while no staining was present in the sclera. Calbindin 2 was detected using immunohistochemistry and Western blotting in the corneal epithelium, endothelium and stroma. qRT-PCR confirmed its expression in epithelial and endothelial cells.Three proteins expressed constitutively in mesothelial cells were detected in the human cornea. The possible function of mesothelin in cell-cell contact on the ocular surface is discussed. The presence of HBME-1 protein in the endothelial layer may indicate a still unknown function that could be shared with mesothelial cells of the pleura and peritoneum. The much more pronounced occurrence of calbindin 2 in the corneal epithelium compared to fewer positive endothelial cells explains the higher turnover of epithelial cells compared to the proliferatively inactive endothelium.     

Evaluation of potential organ culture media for eye banking using human donor corneas

AIM: To evaluate the ability of different commercially available cell culture solutions to preserve human donor corneas during 3 weeks of "closed system" organ culture at physiological temperature. This screening was performed in an attempt to establish a rational basis for the development of a serum-free organ culture medium for eye banking. METHODS: 72 normal human donor corneas were organ cultured for 21 days at 31 degrees C in eight different test media (nine corneas in each group). The basic culture solutions included: minimal essential medium (MEM), MEM with stabilised L-glutamine, M199, DIF-1000, SFM, F99, and F99 with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids (termed F99-Sr). All media were supplemented with 2% fetal calf serum (FCS), except for MEM, which was also studied at 8% FCS. The evaluation parameters included: (1) the endothelial cell loss as evaluated using trypan blue staining; (2) the ability of keratocytes and endothelial cells to incorporate tritiated uridine into RNA as evaluated using autoradiography and digital image analysis; (3) the leakage of immunogenic keratan sulphate as assessed using ELISA; and (4) changes in storage medium pH, glucose, and lactate content. RESULTS: SFM induced the lowest endothelial cell loss of 14% (SD 2%) and the highest RNA synthesis rates of all test solutions supplemented with 2% FCS. Corneas stored in SFM also showed the least leakage of keratan sulphate and the highest glucose consumption and lactate production. In five media (MEM with 2% FCS, MEM with stabilised L-glutamine, M199, F99, and F99-Sr), comparable and intermediate potentials for organ culture were observed with endothelial cell loss of 16-19%. By contrast, 29% (4%) of the endothelium was lost after storage in DIF-1000. Interestingly, the use of 8% FCS (in MEM) had a marked protective effect on the endothelium, which showed the highest RNA synthetic activity combined with a cell loss of only 11% (4%), compared with 19% (6%) at 2% FCS (p<0.05). CONCLUSION: Among the present test solutions, SFM appears to be the most prominent candidate for a new corneal organ culture medium and should be further tested and possibly refined to effectively substitute serum addition.     

Ocular histopathology of systemic mucopolysaccharidosis, type II-A (Hunter syndrome, severe)

A case of mucopolysaccharidosis, Type II-A (Hunter syndrome, severe) is described, with emphasis on ocular ultrastructural findings. Single membrane-bound structures containing fibrillogranular and, less commonly, multi-membranous material were found in conjunctival epithelium, pericytes and fibrocytes; corneal epithelium, keratocytes, and endothelium; trabecular endothelium; iris pigmented epithelium, smooth muscle, and fibrocytes; ciliary pigmented and nonpigmented epithelium and fibrocytes; retinal pigment epithelium and ganglion cells; optic nerve astrocytes and pericytes; and sclerocytes. The most striking accumulation was in the nonpigmented ciliary epithelium. These findings are compared with those seen in MPS II-B, and in other systemic mucopolysaccharidoses. The nature and distribution of inclusions are not specific to any one disorder, but help to signal the presence of one of the storage disorders. Distension of corneal keratocytes may play a role in the corneal clouding seen in some of these disorders. The importance of tissue examination, especially conjunctival biopsy, in the diagnosis of storage disorders and in assessment of future modes of therapy for the mucopolysaccharidoses is discussed.     

Cell death during corneal storage at 4 degrees C.

PURPOSE: To evaluate cell death in human donor corneas stored at 4 degrees C, to determine whether terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labeling (TUNEL) discriminates between apoptosis and necrosis in corneas stored at 4 degrees C. METHODS: Ten human corneas were stored in Optisol (Chiron Ophthalmics, Irvine, CA) at 4 degrees C for periods ranging from 0 to 21 days and then fixed for histologic examination. Central corneal sections from each cornea were examined by transmission electron microscopy (TEM) and by the TUNEL assay. Electron micrographs of at least 15 keratocytes each from the anterior, middle, and posterior stroma were examined by three masked observers who graded each cell as normal, apoptotic, or necrotic. Central sections from the same corneas were processed by the TUNEL assay and evaluated with a laser scanning confocal microscope to determine the percentage of apoptotic cells. RESULTS: By TEM, apoptosis occurred in 23% of the keratocytes and necrosis in 12%. By TUNEL assay, apoptosis occurred in 11% of the keratocytes, with the results in individual corneas being similar to the findings by TEM for apoptosis, rather than for necrosis. By TUNEL assay, apoptosis occurred in 13% of the epithelial cells and in 8% of the endothelial cells. The percentage of apoptotic cells and storage time correlated significantly for the epithelium, but not for the keratocytes or endothelium in this small sample. CONCLUSIONS: Both apoptosis and necrosis occur in cells during corneal storage at 4 degrees C, with apoptosis appearing to predominate. The TUNEL assay identifies cells undergoing apoptosis, but not necrosis, in corneal tissue. Inhibition of apoptosis in corneas stored at 4 degrees C may prolong acceptable storage times.     

Confocal microscopic evidence of increased Langerhans cell activity after corneal metal foreign body removal

PURPOSE: The purpose of the study was to examine the corneal inflammatory reaction and Langerhans cells with confocal microscopy after metal foreign body removal. METHODS: Corneal metal foreign body was removed from 9 eyes of 9 consecutive patients 12.1+/-13.6 (4 to 72) hours after superficial angle grinder injury. Both eyes were examined with the Heidelberg Retina Tomograph II (HRT II) Rostock Cornea Module. Morphology and density of epithelium, nerves, metal deposits, keratocytes, endothelium, and Langerhans cells were compared to the uninjured fellow eyes (controls). RESULTS: Irregularity and partially missing superficial epithelium was found in all cases. Around the area of injury prolonged basal and wing epithelial cells were found in all eyes. The basal epithelium density is lower than in the control eye (p=0.043). Density of Langerhans cells (68.1+/-24.2/mm2) was increased in the epithelium, compared to controls (35.2+/-21.8/mm2, p=0.012). Keratocyte and endothelium densities were not different from that of controls. Some keratocytes showed signs of activation and the inhomogeneous background reflectivity revealed extracellular matrix alterations. Inflammatory reaction was observed up to260 micronm depth. The metal foreign body particles had high reflectivity and irregular edge. CONCLUSIONS: In vivo confocal microscopy provided additional information to biomicroscopic signs such as epithelial damage and inflammation. It showed the effects of metal foreign bodies in the cornea: nerve damage and Langerhans cell density increase. Langerhans cells seem to play an important role in the inflammatory response after corneal foreign body injuries.     

角膜不同组织免疫原性分析

目的：了解角膜3层组织的免疫原性相对值。方法：（1）细胞免疫：将C57BL-6小鼠脊部皮下组织分离成囊袋,分别异位植入异种角膜3层组织,于术后12d用流式细胞仪检测鼠血清中抗CD25和抗CD71mAb标记活化的T淋巴细胞。（2）体液免疫：分别用异种角膜上皮细胞和全角膜匀浆免疫动物获取抗血清,以酶联免疫吸附法测定含细胞的3层角膜组织的免疫原性。结果：（1）细胞免疫：按等组织厚度计算,角膜内皮、上皮及基质组织各占角膜总免疫原性的比率分别为70.75%、27.63%及1.62%。（2）体液免疫：角膜内皮的免疫原性最强,角膜上皮次之,角膜基质最弱,分别约占整个角膜免疫原性的62.11%、31.77%及6.12。结论：3层角膜组织中角膜内皮免疫原性最强,基质免疫原性最弱。     

Differential regulation of fibronectin synthesis in three different types of corneal cells

The production of fibronectin (FN) and its response to serum or epidermal growth factor (EGF) were investigated in three different types of rabbit corneal cells cultured in vitro. The corneal epithelial cells, stromal fibroblasts (keratocytes) and endothelial cells were separately cultured in different media: basic medium containing minimal serum (0.5%), basic medium with supplementary serum at a final concentration of 10% and basic medium with 100 ng/ml EGF, respectively. FN production by each type of cell was examined either by the immunofluorescent staining method or by the metabolic labeling method followed by immunoprecipitation of FN in the culture medium. Each type of corneal cell produced and secreted FN. FN secretion into the culture medium by keratocytes and by endothelial cells was enhanced by the addition of EGF. However, FN secretion by epithelial cells was lowered by the additional serum or EGF. Furthermore, when the epithelial cells were cultured in the basic medium, DNA synthesis was low but FN secretion was high. These results suggest that the control mechanism of FN production differs between epithelial cells and keratocytes or endothelial cells.