Full-field Optical Coherence Microscopy for Histology-like Analysis of Stromal Features in Corneal Grafts.

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

Corneal stromal stem cells restore transparency after N2 injury in mice.

Corneal scarring associated with various corneal conditions is a leading cause of blindness worldwide. The present study aimed to test the hypothesis that corneal stromal stem cells have a therapeutic effect and are able to restore the extracellular matrix organization and corneal transparency in vivo. We first developed a mouse model of corneal stromal scar induced by liquid nitrogen (N2 ) application. We then reversed stromal scarring by injecting mouse or human corneal stromal stem cells in injured cornea. To characterize the mouse model developed in this study and the therapeutic effect of corneal stromal stem cells, we used a combination of in vivo (slit lamp, optical coherence tomography, in vivo confocal microscopy, optical coherence tomography shear wave elastography, and optokinetic tracking response) and ex vivo (full field optical coherence microscopy, flow cytometry, transmission electron microscopy, and histology) techniques. The mouse model obtained features early inflammation, keratocyte apoptosis, keratocyte transformation into myofibroblasts, collagen type III synthesis, impaired stromal ultrastructure, corneal stromal haze formation, increased corneal rigidity, and impaired visual acuity. Injection of stromal stem cells in N2 -injured cornea resulted in improved corneal transparency associated with corneal stromal stem cell migration and growth in the recipient stroma, absence of inflammatory response, recipient corneal epithelial cell growth, decreased collagen type III stromal content, restored stromal ultrastructure, decreased stromal haze, decreased corneal rigidity, and improved vision. Our study demonstrates the ability of corneal stromal stem cells to promote regeneration of transparent stromal tissue after corneal scarring induced by liquid nitrogen.

Implementation of artifact-free circular dichroism SHG imaging of collagen.

Second harmonic generation (SHG) enables in situ imaging of fibrillar collagen architecture in connective tissues. Recently, Circular Dichroism SHG (CD-SHG) microscopy has been implemented to take advantage of collagen chirality to improve 3D visualization. It measures the normalized difference in the SHG signal obtained upon excitation by left versus right circular polarizations. However, CD-SHG signal is not well characterized yet, and quite different CD-SHG values are reported in the literature. Here, we identify two major artifacts that may occur in CD-SHG experiments and we demonstrate that thorough optimization and calibration of the experimental setup are required for CD-SHG imaging. Notably it requires a careful calibration of the incident circular polarizations and a perfect mechanical stabilization of the microscope stage. Finally, we successfully record CD-SHG images in human cornea sections and confirm that this technique efficiently reveals collagen fibrils oriented out of the focal plane.

Long-Term Results of Cultured Limbal Stem Cell Versus Limbal Tissue Transplantation in Stage III Limbal Deficiency.

We aimed to evaluate efficiency and safety of transplantation of limbal stem cells (LSC) cultured on human amniotic membrane with no feeders and to compare cultured LSC with limbal tissue transplantation. Thirty eyes with stage III LSC deficiency were treated with autologous (autoLSC) or allogeneic (alloLSC) cultured LSC transplantation (prospective phase II clinical trial; average follow-up time, 72 months) or autologous (autoLT) or allogeneic (alloLT) limbal tissue transplantation (retrospective control group; average follow-up time, 132 months) between 1993 and 2014. The 5-year graft survival defined by absence of recurrence of the clinical signs of limbal deficiency was 71% for autoLSC, 0% for alloLSC, 75% for autoLT, and 33% for alloLT. Visual acuity improved by 9.2 lines for autoLSC and 3.3 lines for autoLT. It decreased by 0.7 lines for alloLSC and 1.9 lines for alloLT. Adverse events were recorded in 1/7 autoLSC, 7/7 alloLSC, 6/8 autoLT, and 8/8 alloLT patients. Corneal epithelial defect was the only adverse event recorded after autoLSC, whereas severe sight-threatening adverse events were recorded in the remaining three groups. Compared with failed grafts, successful grafts featured greater decrease in fluorescein staining, greater superficial vascularization-free corneal area, lower variability of the corneal epithelial thickness, and higher corneal epithelial basal cell density. Autologous cultured LSC transplantation was associated with high long-term survival and dramatic improvement in vision and was very safe. Autologous limbal tissue transplantation resulted in similar efficiency but was less safe. Cadaver allogeneic grafts resulted in low long-term success rate and high prevalence of serious adverse events. Stem Cells Translational Medicine 2019;8:1230&1241.

Stromal striae: a new insight into corneal physiology and mechanics.

We uncover the significance of a previously unappreciated structural feature in corneal stroma, important to its biomechanics. Vogt striae are a known clinical indicator of keratoconus, and consist of dark, vertical lines crossing the corneal depth. However we detected stromal striae in most corneas, not only keratoconus. We observed striae with multiple imaging modalities in 82% of 118 human corneas, with pathology-specific differences. Striae generally depart from anchor points at Descemet's membrane in the posterior stroma obliquely in a V-shape, whereas in keratoconus, striae depart vertically from posterior toward anterior stroma. Optical coherence tomography shear wave elastography showed discontinuity of rigidity, and second harmonic generation and scanning electron microscopies showed undulation of lamellae at striae locations. Striae visibility decreased beyond physiological pressure and increased beyond physiological hydration. Immunohistology revealed striae to predominantly contain collagen VI, lumican and keratocan. The role of these regions of collagen VI linking sets of lamellae may be to absorb increases in intraocular pressure and external shocks.

New parameters in assessment of human donor corneal stroma.

PURPOSE: To provide quantitative parameters for assessment of human donor corneal stroma by imaging stromal features of diseased and normal human corneas with full-field optical coherence microscopy (FFOCM), using confocal microscopy (CM) and histology as reference techniques. METHODS: Bowman's layer (BL) thickness and keratocyte density were assessed ex vivo in 23 human donor corneas and 27 human pathological corneas (keratoconus and other corneal disorders) with FFOCM, CM and histology. Stromal backscattering was assessed with FFOCM. Additionally, 10 normal human corneas were assessed in vivo with CM. RESULTS: In FFOCM, the logarithm of the normalized stromal reflectivity was a linear function of stromal depth (R2  = 0.95) in human donor corneas. Compared with keratoconus corneas, human donor corneas featured higher BL thickness (p = 0.0014) with lower coefficient of variation (BL-COV; p = 0.0002), and linear logarithmic stromal reflectivity with depth (higher R2 , p = 0.0001). Compared with other corneal disorders, human donor corneas featured lower BL-COV (p = 0.012) and higher R2 (p = 0.0001). Using the 95% confidence limits of the human donor cornea group, BL thickness < 6.5 µm (sensitivity, 57%; specificity, 100%), BL-COV > 18.6% (79%; 100%) and R2  < 0.94 (93%; 71%) were revealed as indictors of abnormal cornea. In CM, keratocyte density decreased with stromal depth (r = -0.56). The mean overall keratocyte density (cells/mm2 ) was 205 in human donor corneas, 244 in keratoconus, 176 in other corneal disorders and 386 in normal corneas. CONCLUSION: Full-field optical coherence microscopy (FFOCM) provides precise and reliable parameters for non-invasive assessment of human donor corneal stroma during storage, enabling detection of stromal disorders that could impair the results of keratoplasty.

Imaging Microscopic Features of Keratoconic Corneal Morphology.

PURPOSE: To search for gold-standard histology indicators using alternative imaging modalities in keratoconic corneas. METHODS: Prospective observational case-control study. Fourteen keratoconic corneas and 20 normal corneas (10 in vivo healthy subjects and 10 ex vivo donor corneas) were examined. Images of corneas were taken by spectral domain optical coherence tomography (SD-OCT) and in vivo confocal microscopy (IVCM) before keratoplasty. The same removed corneal buttons were imaged after keratoplasty with full-field optical coherence microscopy (FFOCM) and then fixed and sent for histology. Controls consisted of normal subjects imaged in vivo with IVCM and donor corneas imaged ex vivo with FFOCM. Corneal structural changes related to pathology were noted with each imaging modality. Cell density was quantified by manual cell counting. RESULTS: Keratoconus indicators (ie, epithelial thinning/thickening, cell shape changes, ferritin deposits, basement membrane anomalies, Bowman layer thinning, ruptures, interruptions, scarring, stromal modifications, and appearance of Vogt striae) were generally visible with all modalities. Additional features could be seen with FFOCM in comparison with gold-standard histology, particularly in the Bowman layer region, whereas the combination of SD-OCT plus IVCM detected 76% of those features detected in histology. Three-dimensional FFOCM imaging aided interpretation of two-dimensional IVCM and SD-OCT data. Basal epithelial cell and keratocyte densities were significantly lower in patients with keratoconus than those in normals (P < 0.0001). CONCLUSIONS: Structural and cellular assessment of the keratoconic cornea by means of either in vivo SD-OCT combined with IVCM or ex vivo FFOCM in both cross-sectional and en face views can detect as many keratoconus indicators as gold-standard histology.