Naoyuki Morishige

Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals

PURPOSE: To investigate the feasibility of using femtosecond‐pulse lasers to produce second‐harmonic generated (SHG) signals to noninvasively assess corneal stromal collagen organization. SETTING: The Eye Institute, University of California, Irvine, California, USA. METHODS: Mouse, rabbit, and human corneas were examined by two‐photon confocal microscopy using a variable‐wavelength femtosecond lasers to produce SHG signals. Two types were detected: forward scattered and backward scattered. Wavelength dependence of the SHG signal was confirmed by spectral separation using the 510 Meta (Zeiss). To verify the spatial relation between SHG signals and corneal cells, staining of cytoskeletons and nuclei was performed. RESULTS: Second‐harmonic‐generated signal intensity was strongest with an excitation wavelength of 800 nm for all 3 species. Second‐harmonic‐generated forward signals showed a distinct fibrillar pattern organized into bands suggesting lamellae, while backscattered SHG signals appeared more diffuse and indistinct. Reconstruction of SHG signals showed two patterns of lamellar organization: highly interwoven in the anterior stroma and orthogonally arranged in the posterior stroma. Unique to the human cornea was the presence of transverse, sutural lamellae that inserted into Bowmans layer, suggesting an anchoring function. CONCLUSIONS: Using two‐photon confocal microscopy to generate SHG signals from the corneal collagen provides a powerful new approach to noninvasively study corneal structure. Human corneas had a unique organizational pattern with sutural lamellae to provide important biomechanical support that was not present in mouse or rabbit corneas.

Application of second harmonic imaging microscopy to assess structural changes in optic nerve head structure ex vivo

Glaucoma represents the second leading cause of blindness worldwide. While both age and intraocular pressure (IOP) are well-recognized risk factors for this disease, the underlying pathologic process involves the accelerated death of retinal ganglion cells (RGCs) that is associated with progressive loss of vision. The loss of RGCs has been postulated to occur primarily by injury to axons in the optic nerve head (ONH) due to its anatomic features and the mechanical vulnerability of the lamina cribrosa, the specialized ONH zone comprised of collagen beams that define the channels or pores through which axon bundles exit the eye. Recent advances in multiphoton microscopy using femtosecond lasers that generate second harmonic (SH) signals from collagen allows for direct optical imaging of the lamina cribrosa. We assess the application of SH generated microscopy (SHG) to the study of the ONH, and test the general hypothesis that increasing intraocular pressure in the same eye results in the movement of ONH collagen beams leading to distortion of the lamina cribrosa channels and compression of the axon bundles.

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.

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.

Functional Foxp3+ CD4+ CD25(Bright+) “Natural” Regulatory T Cells Are Abundant in Rabbit Conjunctiva and Suppress Virus-Specific CD4+ and CD8+ Effector T Cells during Ocular Herpes Infection

ABSTRACT We studied the phenotype and distribution of “naturally” occurring CD4+ CD25+ T regulatory cells (CD4+ CD25+ nTreg cells) resident in rabbit conjunctiva, the main T-cell inductive site of the ocular mucosal immune system, and we investigated their suppressive capacities using herpes simplex virus type 1 (HSV-1)-specific effector T (Teff) cells induced during ocular infection. The expression of CD4, CD25, CTLA4, GITR, and Foxp3 was examined by reverse transcription-PCR, Western blotting, and fluorescence-activated cell sorter analysis in CD45+ pan-leukocytes isolated from conjunctiva, spleen, and peripheral blood monocyte cells (PBMC) of HSV-1-infected and uninfected rabbits. Normal conjunctiva showed a higher frequency of CD4+ CD25(Bright+) T cells than did spleen and PBMC. These cells expressed high levels of Foxp3, GITR, and CTLA4 molecules. CD4+ CD25(Bright+) T cells were localized continuously along the upper and lower palpebral and bulbar conjunctiva, throughout the epithelium and substantia propria. Conjunctiva-derived CD4+ CD25(Bright+) T cells, but not CD4+ CD25(low) T cells, efficiently suppressed HSV-specific CD4+ and CD8+ Teff cells. The CD4+ CD25(Bright+) T-cell-mediated suppression was effective on both peripheral blood and conjunctiva infiltrating Teff cells and was cell-cell contact dependent but independent of interleukin-10 and transforming growth factor β. Interestingly, during an ocular herpes infection, there was a selective increase in the frequency and suppressive capacity of Foxp3+ CD4+ CD25(Bright+) T cells in conjunctiva but not in the spleen or in peripheral blood. Altogether, these results provide the first evidence that functional Foxp3+ CD4+ CD25(Bright+) Treg cells accumulate in the conjunctiva. It remains to be determined whether conjunctiva CD4+ CD25+ nTreg cells affect the topical/mucosal delivery of subunit vaccines that stimulate the ocular mucosal immune system.

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.

Persistent epithelial defects due to neurotrophic keratopathy treated with a substance p-derived peptide and insulin-like growth factor 1.

PurposeThe loss of corneal sensation results in the development of persistent corneal epithelial defects. The combination of a substance P-derived peptide, phenylalanine -glycine-leucine-methionine (FGLM)-amide, and insulin-like growth factor 1 (IGF-1) stimulates corneal epithelial migration in vitro and corneal epithelial wound closure in vivo. The clinical efficacy of eye drops containing FGLM-amide and IGF-1 for the treatment of persistent epithelial defects in individuals with neurotrophic keratopathy was examined in a prospective open study.MethodsEleven patients with persistent corneal epithelial defects associated with neurotrophic keratopathy were treated for up to 28 days by the administration of eye drops containing FGLM-amide and IGF-1. The course of epithelial healing was monitored by slit-lamp examination, and visual acuity was measured before and after treatment.ResultsComplete epithelial resurfacing was achieved in eight of the nine (89%) cases of persistent epithelial defects in the nine patients who received the full course of treatment. Epithelial defects had completely resurfaced in two of nine (22%) and five of nine (56%) cases within 1 and 2 weeks, respectively, after treatment initiation. No adverse effects of treatment were observed in any of the 11 patients.ConclusionsEye drops containing FGLM-amide and IGF-1 induced the rapid resurfacing of persistent epithelial defects in individuals with neurotrophic keratopathy. Jpn J Ophthalmol 2007;51:442–447

Abnormal light scattering detected by confocal biomicroscopy at the corneal epithelial basement membrane of subjects with Type II diabetes

Aims/hypothesis. Abnormalities of the basement membrane are thought to contribute to the complications of diabetes. The suitability of the cornea for detecting such abnormalities was assessed by determining its light-scattering index, a quantitative measure of tissue reflectivity in the basement membrane zone, with a confocal biomicroscope. Methods. The light-scattering index was measured in 65 subjects with Type II (non-insulin-dependent) diabetes mellitus and 18 control subjects and was evaluated for its possible relation to the stage of diabetic retinopathy. Diabetic retinopathy was staged by ophthalmoscopic examination as non-diabetic (NDR), simple (SDR), preproliferative (PPDR), or proliferative (PDR). Results. Examination of the cornea layer-by-layer with a confocal biomicroscope did not show any marked differences in morphology between diabetic and control subjects. The LSI (mean ± SD) was 0.81 ± 0.13, 0.87 ± 0.09, 0.90 ± 0.09, 0.90 ± 0.13, and 1.02 ± 0.25 in control subjects and in diabetic subjects with NDR, SDR, PPDR, or PDR, respectively; the light-scattering index of diabetic subjects with PDR was significantly greater than that of the control subjects (p = 0.001). An LSI greater than 1.0 was detected in 5.6, 6.3, 15.0, 15.4, and 50.0 % of control subjects and of patients with NDR, SDR, PPDR, or PDR, respectively; the percentage of subjects with an LSI greater than 1.0 was significantly increased in diabetic patients with PDR than for control subjects. Conclusion/interpretation. These results suggest that the LSI increases with the stage of diabetic retinopathy, and that measurement of corneal light scattering could provide an index of basement membrane abnormality in people with diabetes. [Diabetologia (2001) 44: 340–345]