Tai-ichiro Chikama

Differential distribution of subchains of the basement membrane components type IV collagen and laminin among the amniotic membrane, cornea, and conjunctiva.

PURPOSE Amniotic membrane transplantation has been reported to be an effective surgical procedure for the reconstruction of the anterior segment of the eye. To understand better the function of transplanted amniotic membrane, we compared the distributions of subchains of type IV collagen and laminin in the amniotic membrane to those in the cornea and conjunctiva. METHODS Five human corneas with conjunctivas and three human amniotic membranes were frozen in OCT compound. Cryosections were cut with a cryostat and stained by an indirect immunofluorescent microscopy. We used antibodies of the collagen alpha2(IV) and alpha5(IV) subchains, laminin-1, laminin-5, fibronectin, and type VII collagen. RESULTS In the conjunctival basement membrane and the amniotic membrane, fluorescence was evident for collagen alpha2(IV) but not for collagen alpha5(IV). By contrast, in the corneal basement membrane, fluorescence was apparent for the collagen alpha5(IV) subchain but not for the collagen alpha2(IV) subchain. Laminin-1, laminin-5, fibronectin, and type VII collagen were present in all the basement membranes examined. CONCLUSION The distribution of alpha subchains of type IV collagen in the amniotic membrane was identical to that in the conjunctiva but different from that in the cornea. No difference in the distribution pattern of other basement membrane components was observed. These results demonstrate that the basement membrane of the amniotic membrane and the conjunctiva might share the same components; therefore, the amniotic membrane might be useful as a replacement for the basement membrane of the conjunctiva.

Cell–matrix and cell–cell interactions during corneal epithelial wound healing

The corneal epithelium serves as a barrier and contributes to the maintenance of corneal transparency and rigidity. In most instances, corneal epithelial defects caused by simple injury are resurfaced promptly. However, in individuals with certain clinical conditions, such as herpes simplex virus infection, neurotrophic keratopathy or diabetic keratopathy, corneal epithelial defects persist and do not respond to conventional treatment regimens because of delayed epithelial wound healing. After the corneal epithelium is removed by injury, the remaining epithelial cells migrate over the denuded surface of the cornea in a manner that is dependent both on the interaction of the cells with the underlying substrate and on cell-cell adhesion. In this review, we describe the specific roles of cell-matrix and cell-cell interactions during the course of corneal epithelial wound healing. The clinical implications of the basic research findings are also discussed.

Correlation of corneal sensation, but not of basal or reflex tear secretion, with the stage of diabetic retinopathy.

Purpose. To examine the possible relation between corneal sensation or tear secretion and the stage of diabetic retinopathy in diabetic patients. Methods. Total reflex or basal tear secretion and corneal sensation were determined in 95 patients with type II diabetes mellitus and 58 nondiabetic control subjects. Tear secretion was measured by the Schirmer test and corneal sensation with a Cochet-Bonnet esthesiometer. Results. Corneal sensation and total or reflex tear secretion were significantly reduced in diabetic patients compared with nondiabetic controls. The loss of corneal sensation, but not that of tear secretion, was significantly correlated with stage of diabetic retinopathy in diabetic patients who were diagnosed with no diabetic retinopathy, simple diabetic retinopathy, preproliferative retinopathy, or proliferative retinopathy. Conclusion. Both corneal sensation and total or reflex tear secretion are reduced in individuals with diabetes. The decrease in corneal sensation, but not that in each tear secretion, was correlated with the stage of diabetic retinopathy. Given that loss of corneal sensation is a manifestation of diabetic polyneuropathy, these results are consistent with the notion that both diabetic retinopathy and polyneuropathy result from a basement membrane abnormality.

Comparison of confocal biomicroscopy and noncontact specular microscopy for evaluation of the corneal endothelium.

Purpose. To compare the clinical efficacy of confocal biomicroscopy with that of noncontact specular microscopy for the evaluation of the corneal endothelium. Methods. The corneal endothelium was examined in 14 normal subjects (28 eyes) and in 6 patients (11 eyes) with Fuchs corneal endothelial dystrophy using a noncontact specular microscope (SP-2000P, Topcon, Japan) and a confocal biomicroscope (ConfoScan, Tomey, Japan). The images and the calculated densities of corneal endothelial cells obtained by the 2 techniques were compared. Results. For normal subjects, the images of corneal endothelial cells obtained by the 2 techniques were almost identical, although the density of these cells determined by confocal biomicroscopy (2916 ± 334 cells/mm2) was slightly higher than that determined by noncontact specular microscopy (2765 ± 323 cells/mm2). In contrast, whereas clear images of corneal endothelial cells, allowing the determination of cell density, were obtained for all 11 eyes of the patient group by confocal biomicroscopy, clear images were obtained for only 4 of these 11 eyes (36.4%) by noncontact specular microscopy. Conclusion. Both noncontact specular microscopy and confocal biomicroscopy revealed the shapes and number of endothelial cells in the normal cornea. However, for corneas with Fuchs dystrophy, clear images were obtained only by confocal biomicroscopy. Confocal biomicroscopy is thus an effective tool for evaluation of the diseased corneal endothelium.

Open clinical study of eye-drops containing tetrapeptides derived from substance P and insulin-like growth factor-1 for treatment of persistent corneal epithelial defects associated with neurotrophic keratopathy

Background/aims: Loss of corneal sensation results in the development of persistent corneal epithelial defects. The combination of a substance P-derived peptide (FGLM-amide) and an insulin-like growth factor-1 (IGF-1)-derived peptide (SSSR) stimulates rabbit corneal epithelial migration in vitro and rabbit corneal epithelial wound closure in vivo. The clinical efficacy of eye-drops containing FGLM-amide and SSSR for the treatment of persistent corneal epithelial defects in individuals with neurotrophic keratopathy was examined in a prospective open study. Methods: Twenty-five consecutive patients (26 eyes) with persistent corneal epithelial defects associated with neurotrophic keratopathy were treated by administration of eye-drops containing FGLM-amide and SSSR. The course of epithelial healing was monitored by slit-lamp examination. Results: Epithelial defects resurfaced completely in 19 of the 26 eyes (73%) within 4 weeks after treatment initiation. Complete resurfacing of epithelial defects was apparent in 18 of 22 (82%) or in one of four (25%) eyes without or with limbal stem cell deficiency, respectively. No adverse effects of treatment were observed in any subject. Conclusion: Eye-drops containing FGLM-amide and SSSR induced the rapid resurfacing of persistent epithelial defects in stem cell-positive individuals with neurotrophic keratopathy.

Cytokine, Chemokine, and Adhesion Molecule Expression Mediated by MAPKs in Human Corneal Fibroblasts Exposed to Poly(I:C)

PURPOSE Polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, interacts with Toll-like receptor (TLR)-3 and thereby elicits immunoinflammatory responses characteristic of viral infection. The effects of poly(I:C) on the expression of proinflammatory cytokines, chemokines, and adhesion molecules, as well as the signaling pathways that underlie such effects, were investigated in cultured human corneal fibroblasts. METHODS Expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was evaluated by immunoblot and immunofluorescence analyses. Release of the proinflammatory cytokine IL-6 and of the chemokines interleukin (IL)-8, granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, eotaxin, and RANTES was measured with assay kits. Subcellular localization of the p65 subunit of the transcription factor nuclear factor (NF-kappaB) was examined by immunofluorescence analysis. Expression of TLR3, phosphorylation (activation) of mitogen-activated protein kinases (MAPKs), and phosphorylation and degradation of the NF-kappaB-inhibitory protein IkappaB-alpha was assessed by immunoblot analysis. RESULTS Poly(I:C) induced the up-regulation of TLR3, the release of IL-6, IL-8, G-CSF, MIP-1beta, eotaxin, and RANTES, and the expression of ICAM-1 and VCAM-1 in corneal fibroblasts. It also activated the MAPKs ERK, p38, and JNK and induced the phosphorylation and degradation of IkappaB-alpha and the nuclear translocation of p65 in these cells. Poly(I:C)-induced expression of IL-6, IL-8, G-CSF, MIP-1beta, exotaxin, RANTES, and ICAM-1 was inhibited differentially by the MAPK inhibitors PD98059 and SB203580 and by JNK inhibitor II. CONCLUSIONS Poly(I:C) induces the up-regulation of TLR3, the MAPK-dependent expression of proinflammatory cytokines, chemokines, and adhesion molecules and the activation of NF-kappaB in human corneal fibroblasts. Corneal fibroblasts may thus play an important role in the modulation of local immune and inflammatory responses to viral infection in the corneal stroma.

Detection of Subepithelial Fibrosis Associated with Corneal Stromal Edema by Second Harmonic Generation Imaging Microscopy

PURPOSE Human corneas with or without stromal edema were examined by second harmonic generation (SHG) imaging microscopy to characterize stromal collagen organization. METHODS Tissue buttons from 31 corneas with stromal edema and 8 normal corneas were fixed, and 3-mm(2) blocks were cut and stained with phalloidin, to visualize the cytoskeleton. The blocks were examined by SHG imaging with a laser confocal microscope and a mode-locked titanium:sapphire femtosecond laser. Samples were scanned to a depth of 150 microm from the surface of Bowmans layer, and SHG forward- and backscatter signals were collected. Phalloidin staining was detected by conventional laser confocal microscopy. The three-dimensional structure of the anterior segment of the cornea was reconstructed from stacked SHG images. RESULTS Three-dimensional reconstruction of SHG signals showed adherence of interwoven collagen lamellae in the anterior stroma to Bowmans layer in both normal and edematous corneas. Abnormal SHG signals at the level of Bowmans layer were observed in edematous corneas; three-dimensional images revealed that these signals were actually localized above Bowmans layer and were indicative of subepithelial fibrosis. Phalloidin staining showed transdifferentiation of stromal cells into fibroblastic cells in edematous corneas. The incidence of subepithelial fibrosis or of fibroblastic cells increased beginning 12 months after the onset of clinical stromal edema. CONCLUSIONS SHG imaging of the anterior segment of edematous corneas revealed a normal appearance of interwoven collagen lamellae in the anterior stroma. The development of subepithelial fibrosis beginning 12 months after the onset of edema suggests that stromal edema may be a progressive disease.

The NK1 receptor and its participation in the synergistic enhancement of corneal epithelial migration by substance P and insulin‐like growth factor‐1

1 We have previously shown that substance P (SP) and insulin‐like growth factor‐1 (IGF‐1) act synergistically to enhance the migration of rabbit corneal epithelial cells in an organ culture model. The present study was designed to identify the epithelial cell SP receptor that participates in this synergistic effect. 2 Rabbit corneal blocks were incubated for 24 h, then the length of the path of epithelial migration was measured. Reagents tried in the TC‐199 culture medium, in the presence or absence of IGF‐1, were: SP, agonists of tachykinin receptors NK1; NK2or NK3 and antagonists of tachykinin receptors NK1 or NK2. 3 The binding characteristics of SP receptors were examined in rabbit cultured corneal epithelial cells by binding assays with [125I]‐SP in the presence or absence of excess unlabelled SP or ligands of NKl, NK2 or NK3 receptors. 4 As was demonstrated previously, SP and IGF‐1 stimulated epithelial migration when they were added to the culture medium together, but individually they had no effect. NK1 agonists had the same synergistic effect with IGF‐1 as did SP, but the NK2 and NK3 agonists did not. Furthermore, the NK1 antagonist abolished the synergistic effect of SP and IGF‐1, but the NK2 antagonist had no effect. 5 SP bound specifically to rabbit cultured corneal epithelial cells. The binding affinity was 0.44 nM and there were 2.43 × 104 binding sites per cell. The NK1 ligand competed, in a dose‐dependent fashion, with the binding of SP to corneal epithelial cells, but neither the NK2 nor NK3 ligand affected binding. 6 We conclude that the SP receptor in rabbit corneal epithelial cells is NK1 and that this receptor participates in the synergistic enhancement of corneal epithelial migration by SP and IGF‐1. The precise mechanism(s) of this interaction requires more study. These findings imply that both neural and humoral factors are essential for the maintenance and healing of corneal epithelium.

Synergistic effect with Phe‐Gly‐Leu‐Met‐NH2 of the C‐terminal of substance P and insulin‐like growth factor‐1 on epithelial wound healing of rabbit cornea

We previously reported that substance P and insulin‐like growth factor‐1 (IGF‐1) synergistically stimulate corneal epithelial wound healing in vitro and in vivo. We wished to identify which portion of the amino acid sequence of substance P might be responsible for this synergism. Corneal epithelial migration was not affected by the addition of any one of the following factors: substance P; Phe‐Gly‐Leu‐Met‐NH2 (C‐terminal of substance P); Val‐Gly‐Leu‐Met‐NH2 (C‐terminal of neurokinin A, neurokinin B, and kassinin); Tyr‐Gly‐Leu‐Met‐NH2 (C‐terminal of physalaemin); Ile‐Gly‐Leu‐Met‐NH2 (C‐terminal of eledoisin); or Gly‐Leu‐Met‐NH2 (common C‐terminal of tachykinins). In the presence of IGF‐1, only substance P and Phe‐Gly‐Leu‐Met‐NH2 were synergistic in stimulating corneal epithelial migration in a dose‐dependent fashion. The combination of Phe‐Gly‐Leu‐Met‐NH2 and IGF‐1 did not affect the incorporation of [3H]‐thymidine into corneal epithelial cells. Treatment with Phe‐Gly‐Leu‐Met‐NH2 and IGF‐1, but not with Phe‐Gly‐Leu‐Met‐NH2 or IGF‐1 alone, increased attachment of corneal epithelial cells to a fibronectin matrix. The levels of α5 and β1 integrin were not affected by Phe‐Gly‐Leu‐Met‐NH2 or IGF‐1 alone, but they were significantly increased by the combination of Phe‐Gly‐Leu‐Met‐NH2 and IGF‐1. Topical application of the same combination facilitated corneal epithelial wound closure in vivo. These results demonstrated that Phe‐Gly‐Leu‐Met‐NH2, a sequence of 4 amino‐acids of the C‐terminal of substance P, is the minimum sequence necessary to produce the synergistic effects of substance P and IGF‐1 on corneal epithelial wound healing.

Three-Dimensional Analysis of Collagen Lamellae in the Anterior Stroma of the Human Cornea Visualized by Second Harmonic Generation Imaging Microscopy

PURPOSE The structure of collagen lamellae in the anterior stroma of the human cornea is thought to be an important determinant of corneal rigidity. The three-dimensional structure of such collagen lamellae in normal human corneas was examined. METHODS The anterior portion of 27 normal human corneas was obtained from donor tissue for Descemets stripping automated endothelial keratoplasty (DSAEK) surgery, and blocks (∼3-mm square) of the central cornea were examined by second harmonic generation (SHG) imaging microscopy. Each cornea was scanned from the surface of Bowmans layer to a depth of 150 μm, and SHG forward signals were collected. The angles of collagen lamellae immediately below to a depth of 30 μm below Bowmans layer (sutural lamellae) as well as of those at a depth of 50 or 100 μm were measured. The density and width of sutural lamellae were also evaluated. RESULTS Collagen lamellae in the anterior stroma were evenly distributed and randomly oriented. The angle of sutural lamellae relative to Bowmans layer was 19.19 ± 4.34° (mean ± SD). The angles of collagen lamellae at depths of 50 or 100 μm were 8.91 ± 2.91 and 6.91 ± 2.11°, respectively. The density of sutural lamellae was 910.0 ± 480.4/mm(2), and their width was 13.14 ± 5.03 and 7.11 ± 3.00 μm in the region immediately beneath and 30 μm below Bowmans layer, respectively. CONCLUSIONS Collagen lamellae in the anterior stroma of the normal human cornea are interwoven in three dimensions and adhere densely to Bowmans layer. This structure may contribute to the rigidity and curvature of the anterior portion of the cornea.