Jean-Marc Legeais

Multimodal Nonlinear Imaging of the Human Cornea

PURPOSE To evaluate the potential of third-harmonic generation (THG) microscopy combined with second-harmonic generation (SHG) and two-photon excited fluorescence (2PEF) microscopies for visualizing the microstructure of the human cornea and trabecular meshwork based on their intrinsic nonlinear properties. METHODS Fresh human corneal buttons and corneoscleral discs from an eye bank were observed under a multiphoton microscope incorporating a titanium-sapphire laser and an optical parametric oscillator for the excitation, and equipped with detection channels in the forward and backward directions. RESULTS Original contrast mechanisms of THG signals in cornea with physiological relevance were elucidated. THG microscopy with circular incident polarization detected microscopic anisotropy and revealed the stacking and distribution of stromal collagen lamellae. THG imaging with linear incident polarization also revealed cellular and anchoring structures with micrometer resolution. In edematous tissue, a strong THG signal around cells indicated the local presence of water. Additionally, SHG signals reflected the distribution of fibrillar collagen, and 2PEF imaging revealed the elastic component of the trabecular meshwork and the fluorescence of metabolically active cells. CONCLUSIONS The combined imaging modalities of THG, SHG, and 2PEF provide key information about the physiological state and microstructure of the anterior segment over its entire thickness with remarkable contrast and specificity. This imaging method should prove particularly useful for assessing glaucoma and corneal physiopathologies.

A second generation of artificial cornea (Biokpro II)

The properties of a new second-generation colonizable artificial cornea were evaluated in humans. The prosthesis consisted of a peripheral rim of a translucent microporous fluorocarbon polymer (expanded polytetrafluoroethylene) fused with the polymer of the central optic. The optic was made of medical-grade polydimethylsiloxane coated with polyvinylpyrrolidone. Its refractive power was 42.2 diopters and it measured 7.0 mm in diameter and 0.55 mm in thickness. The geometry of the optic was tested by high-frequency ultrasound and intraocular pressure and distensibility were measured in an artificial chamber. Prostheses were implanted in one eye of 13 humans. The average follow-up was 6 months (range 3-9 months). Most of the eyes (11/13) were clinically stable after a 7 months follow up. Seven patients had visual acuity improvements. Mean corrected final visual acuity was 20/200 (range, 20/30 to light perception). Five anatomical failures occured (two extrusions, two retroprosthetic membranes, one endophthalmitis). The new optical core, junction, and surface properties of the polymers offer many advantages, quicker colonization of the supporting skirt, and an optical core with a geometry similar to that of a normal human cornea. Epithelial cells did not migrate over the interface and optical core. It seems that formation of an epithelium over the artificial device is essential for the long-term stability of the implant.

A new mutation (A546T) of the βig-h3 gene responsible for a French lattice corneal dystrophy type IIIA

PURPOSE To characterize the betaig-h3 gene defect in a French family affected with lattice corneal dystrophy type IIIA (LCDIIIA). METHODS Histologic examination was performed from corneal buttons of two patients. Genomic DNA was extracted from leukocytes, and exons of the betaig-h3 gene were amplified by polymerase chain reaction to be directly sequenced. RESULTS Numerous deposits were evident in the stroma and beneath the Bowman membrane, which had all the features of amyloid deposits. Analysis of exon 12 revealed a heterozygous G to A transition on codon 546. CONCLUSION In contrast to Japanese patients, these French patients affected with LCDIIIA carry a distinct mutation of the betaig-h3 gene (A546T instead of P501T). Therefore, it is unclear whether different mutations could result in the same dystrophy or whether we are dealing with clinical heterogeneity of LCDIIIA.

Average 3-Dimensional Models for the Comparison of Orbscan II and Pentacam Pachymetry Maps in Normal Corneas

PURPOSE To assess the reliability of Orbscan (Bausch & Lomb, Salt Lake City, UT) and Pentacam (Oculus, Wetzlar, Germany) central corneal thickness (CCT) and peripheral corneal thickness (PCT) measurements based on 2 methodologies. DESIGN Evaluation of a diagnostic technology. PARTICIPANTS Thirty healthy volunteers were recruited prospectively at the Department of Ophthalmology of the Hôtel-Dieu Hospital, Paris, France. METHODS Central corneal thickness and PCT were assessed, using ultrasound pachymetry (USP) as the gold standard. Two methodologies were used: (1) the traditional analysis of pachymetry data from 1 central and 8 peripheral reference positions on the cornea, and (2) a 3-dimensional (3-D) analysis based on average corneal pachymetry maps constructed for each system (Orbscan, Pentacam, and USP), each operator (operators 1 and 2), and each visit (visits A and B). MAIN OUTCOME MEASURES Repeatability, intersystem reproducibility, interoperator reproducibility, reproducibility over time, and accuracy of Orbscan and Pentacam CCT and PCT measurements. Distribution and statistical significance of the differences between 3-D average maps. RESULTS Repeatability (Orbscan intraclass correlation coefficients [ICCs], 0.967-0.992; Pentacam ICCs, 0.986-0.997), interoperator reproducibility, and reproducibility over time (ICCs, 0.976-0.997) were excellent to almost perfect for both systems. Intersystem agreement was almost perfect for CCT (ICC, 0.980), but less strong for PCT (ICCs, 0.928-0.979). Despite a good to excellent agreement between the optical systems and USP (ICCs, 0.608-0.958), USP CCT readings were thicker (mean difference, up to 15.2 microm; P<0.05), and USP PCT readings were thinner (P<0.05). Orbscan and Pentacam average maps allowed comprehensive interpretation of differences between populations according to the magnitude, distribution, and statistical significance, minimizing the risk of giving excessive weight to few data measured at specific locations on the cornea. CONCLUSIONS Both methodologies showed that Orbscan and Pentacam CCT readings are interchangeable, whereas caution should be used for PCT readings. Interchangeability with USP measurements also was shown to be limited. The high repeatability, interoperator reproducibility, reproducibility over time, the extent of the information generated by a single capture, and the noncontact nature of the Orbscan and Pentacam all suggest that optical systems eventually may replace USP as the gold standard for corneal pachymetry.

Ultrashort pulse laser surgery of the cornea and the sclera

The strongly localized interaction process of ultrashort laser pulses with tissue makes femtosecond lasers a powerful tool for eye surgery. These lasers are now routinely used in refractive surgery and other forms of surgery of the anterior segment of the eye. Several clinical laser systems also offer options for corneal grafting and the potential use of ultrashort pulse lasers in glaucoma surgery has been the object of several recent studies which have shown promising results. While devices aimed for interventions in clear tissue may be based on available solid state or fibre laser technology, the development of tools for surgery in more strongly scattering tissue has to account for the compromised tissular transparency and requires the development of optimized laser sources. The present paper focuses on surgery of clear and pathological cornea as well as sclera. It aims to give an overview over typical medical indications for ultrashort pulse laser surgery, the optics of the tissues involved, the available laser technology, the laser–tissue interaction process, and possible future developments.

A New Fluorocarbon for Keratoprosthesis

Previous studies have demonstrated the potential use of microporous, biocompatible materials to improve the long-term stability of keratoprosthesis. To determine the factors that will influence corneal tissue ingrowth into biocompatible, microporous materials, we have compared three types of fluorocarbon polymers—Impra, Gore-Tex, and Proplast—after intrastromal implantation in rabbit corneas. Despite similar physicochemical structures, a great difference was observed in histologic and ultrastructural cross sections after 4- and 8-month followups. For Gore-Tex, we observed extrusion of the implant and infiltration of necrotic and inflammatory cells. All implants of Proplast also led to significant corneal damage resulting in extrusion of the material. Through the use of electron and light microscopy and image analysis, this study demonstrates the presence of cell differentiation and collagen synthesis in the pores of the Impra implant. Apart from biocompatibility, this experiment demonstrates the influence of pore size, porous microorganization, and biomechanical factors on prosthetic corneal material. Only Impra offers satisfactory interface, allowing fibroblastic cells and neocollagen synthesis into its pores, and it can become transparent

Morphologic and electroretinographic phenotype of SR-BI knockout mice after a long-term atherogenic diet.

PURPOSE To evaluate functional and ultrastructural changes in the retina of scavenger receptor B1 (SR-BI) knockout (KO) mice consuming a high fat cholate (HFC) diet. METHODS Three-month-old male KO and wild-type (WT) mice were fed an HFC diet for 30 weeks. After diet supplementation, plasma cholesterol levels and electroretinograms were analyzed. Neutral lipids were detected with oil red O, and immunohistochemistry was performed on cryostat ocular tissue sections. The retina, Bruchs membrane (BM), retinal pigment epithelium (RPE), and choriocapillaris (CC) were analyzed by transmission electron microscopy. RESULTS Using the WT for reference, ultrastructural changes were recorded in HFC-fed SR-BI KO mice, including lipid inclusions, a patchy disorganization of the photoreceptor outer segment (POS) and the outer nuclear layer (ONL), and BM thickening with sparse sub-RPE deposits. Within the CC, there was abnormal disorganization of collagen fibers localized in ectopic sites with sparse and large vacuolization associated with infiltration of macrophages in the subretinal space, reflecting local inflammation. These lesions were associated with electroretinographic abnormalities, particularly increasing implicit time in a- and b-wave scotopic responses. Abnormal vascular endothelial growth factor (VEGF) staining was detected in the outer nuclear layer. CONCLUSIONS HFC-fed SR-BI KO mice thus presented sub-RPE lipid-rich deposits and functional and morphologic alterations similar to some features observed in dry AMD. The findings lend further support to the hypothesis that atherosclerosis causes retinal and subretinal damage that increases susceptibility to some forms of AMD.

In vivo study of a fluorocarbon polymer-coated intraocular lens in a rabbit model.

Purpose: To evaluate the biocompatibility in rabbit eyes of poly(methyl methacrylate) (PMMA) intraocular lenses (IOLs) that were surface modified using Teflon AF. Setting: Hótel‐Dieu Hospital, Paris Cedex, France. Methods: The IOLs were coated with Teflon AF, an amorphous, transparent, and highly hydrophobic fluorocarbon polymer, by immersing them in Teflon AF 5% and evaporating the solvent (C8F18). The surface quality of the Teflon‐coated IOLs was evaluated by scanning electron microscopy (SEM). Teflon‐coated (n = 20) and control PMMA (n = 10) IOLs were implanted in rabbit eyes. The presence of irisIOL synechias and the number of deposits on the IOL surfaces were clinically evaluated in both groups to assess the antiadhesive effect of Teflon AF. The Tefloncoated IOLs were removed, their surfaces were evaluated by SEM, and their elemental composition was checked by EDXA and Raman spectrometry. Results: The PMMA IOLs were completely coated with Teflon AF. The Teflon group had no iris‐10L synechias and the control group, two extensive synechias. There were significantly fewer deposits on the surfaces of Teflon‐coated IOLs than on the control IOLs 30 and 60 days postoperatively (P < .0001). Scanning electron microscopy showed lens epithelium proliferation and spindle‐shaped cells on the surfaces of the PMMA IOLs and cell deposits on the irregular regions of the Tefloncoated IOLs. White‐yellow spots were present on the surfaces of both IOL types. The elemental composition of Teflon‐coated IOLs was stable. Conclusion: Teflon AF had an antiadhesive effect that increased the biocompatibility of PMMA IOLs in vivo.

In situ monitoring of second-harmonic generation in human corneas to compensate for femtosecond laser pulse attenuation in keratoplasty

The application of femtosecond lasers in corneal transplant surgery requires high pulse energies to compensate for the strong optical scattering in pathological corneas. However, excessive energies deteriorate the quality of the incisions. The aim of this study is to demonstrate the dependence of side effects on local radiant exposure, numerical aperture, and tissue properties, to quantify the penetration depth of the laser for individual corneas, and to provide a method for optimizing the energy in the volume of the cornea. We examine histological and ultrastructural sections of clear and edematous corneas with perforating and lamellar incisions performed at different pulse energies. We demonstrate that the augmented energies in edematous corneas may result in unwanted side effects even when using high numerical apertures. The dependence of the laser beam penetration depth on pulse energy is evaluated by histology and an exponential decrease is observed. We show that the penetration length can be determined by evaluating the backscattered second-harmonic emission associated with the nonlinear optical properties of the tissue. This approach represents a noninvasive method for the in situ quantification of the laser beam attenuation, enabling us to adapt the pulse energy accordingly. Experiments using adapted energies show that the side effects are minimized.

Toxicity of Xylocaine to rabbit corneal endothelium

Purpose: To assess the toxicity of lidocaine hydrochloride (Xylocaine®) to the corneal endothelium. Setting: Department of Ophthalmology, Hotel‐Dieu Hospital, Paris, France. Methods: Rabbit corneas were excised and the endothelium was exposed to balanced salt solution (BSS®), Xylocaine 1 %, or Xylocaine 5% (5 corneas/group) for 20 minutes. The endothelium was then stained with trypan blue and alizarin red, and 5 photomicrographs were taken of each cornea at a standard magnification and analyzed with a digital imaging system (Biocom 200). Results: Xylocaine solutions produced changes in endothelial cell morphology, but there was no cell staining with trypan blue. Corneas exposed to Xylocaine 5% had more marked cell alterations. Small areas of cells were lost from all 15 corneas, mainly at the periphery, but the differences among the 3 groups of corneas were not significant. Conclusion: Exposure of rabbit corneal endothelium to Xylocaine solutions in vitro was not associated with trypan blue staining of endothelial cells.