Christoph Engler

Unfolded protein response in fuchs endothelial corneal dystrophy: a unifying pathogenic pathway?

PURPOSE To assess for activation of the unfolded protein response in corneal endothelium of Fuchs endothelial corneal dystrophy patients. DESIGN Retrospective, comparative case series of laboratory specimens. METHODS Corneal specimens of patients with Fuchs dystrophy and controls with corneal pathologic features other than Fuchs dystrophy were evaluated by transmission electron microscopy (TEM) to evaluate for structural changes of the rough endoplasmic reticulum in corneal endothelium. TEM images were evaluated for alterations of rough endoplasmic reticulum as a sign of unfolded protein response. Normal autopsy eyes, Fuchs dystrophy corneas, and keratoconus corneas were used for immunohistochemistry. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of patient corneas for 3 unfolded protein response markers (GRP78, the alpha subunit of eukaryotic initiation factor 2, C/EBP homologous protein) and 2 apoptosis markers (caspase 3 and 9). Immunohistochemistry signal quantitation of corneal endothelium for evaluation of marker expression was performed using automated software. Corneal sections were assessed quantitatively for levels of immunohistochemistry marker expression. RESULTS TEM showed enlargement of rough endoplasmic reticulum in corneal endothelium of all Fuchs dystrophy specimens. Immunohistochemistry quantitation demonstrated a significant increase in mean signal in corneal endothelium from Fuchs dystrophy patients for markers GRP78, the alpha subunit of eukaryotic initiation factor 2, C/EBP homologous protein, and caspase 9 compared with non-Fuchs dystrophy corneas (P < .05). CONCLUSIONS Results of both TEM and immunohistochemistry indicate activation of unfolded protein response in Fuchs dystrophy. Unfolded protein response activation leads to endothelial cell apoptosis in Fuchs dystrophy and may play a central pathogenic role in this disease.

Transforming Growth Factor-β Signaling Pathway Activation in Keratoconus

PURPOSE To assess the presence of transforming growth factor-β (TGFβ) pathway markers in the epithelium of keratoconus patient corneas. DESIGN Retrospective, comparative case series of laboratory specimens. METHODS Immunohistochemistry results for TGFβ2, total TGFβ, mothers against decacentaplegic homolog (Smad) 2, and phosphorylated Smad2 was performed on formalin-fixed, paraffin-embedded sections of keratoconus patient corneas and normal corneas from human autopsy eyes. Keratoconus patient corneas were divided in two groups, depending on their severity based on keratometer readings and pachymetry. Autopsy controls were age-matched with the keratoconus cases. Immunohistochemistry signal quantification was performed using automated software. Real-time reverse-transcriptase polymerase chain reaction was performed on total ribonucleic acid of epithelium of keratoconus patient corneas and autopsy control corneas. RESULTS Immunohistochemistry quantification showed a significant increase in mean signal in the group of severe keratoconus cases compared with normal corneas for TGFβ2 and phosphorylated Smad2 (P < .05). Immunohistochemistry analysis using antibodies against total TGFβ and Smad2 did not show any significant increase in the keratoconus cases versus the autopsy controls. Reverse-transcriptase polymerase chain reaction exhibited elevated messenger ribonucleic acid levels of Smad2 and TGFβ2 in severe keratoconus corneal epithelium. CONCLUSIONS This work shows increased TGFβ pathway markers in severe keratoconus cases and provides the rationale for investigating TGFβ signaling further in the pathophysiology of keratoconus.

A comprehensive analysis of eye bank-prepared posterior lamellar corneal tissue for use in endothelial keratoplasty.

Purpose: The purpose of this study was to assess eye bank-prepared corneal tissue with regards to the accuracy of postcut tissue thickness, endothelial cell loss, and rate of successful processing. Methods: Details of all 913 corneal tissues processed with an automated microkeratome for use in posterior lamellar transplantation, over a 1-year period, were obtained from a large eye bank. The number and success rate of all attempted cutting procedures were analyzed. The thickness of the corneal button obtained after cutting was compared with the graft thickness requested by the operating surgeon. Changes in endothelial cell density (ECD) during tissue processing were evaluated. Results: The rate of successful tissue preparation increased over the time period examined, from 95% in the first quarter to 99.5% in the fourth quarter. Graft material was frequently slightly thicker than requested by the operating surgeon with 28.3% of tissues cut thicker than requested. Postcut ECD over the entire period increased by an average of 4.7% and was closely related to the starting ECD. Conclusions: There was a very high rate of successful tissue preparation (98.5%), and early failed attempts at tissue cutting were likely the result of the initial learning curve of the involved technicians. Practical considerations resulted in tissue being cut marginally thicker than requested; this is an issue about which the operating surgeon should be aware, because it may possibly influence tissue handling. The quality of the obtained material, as measured by ECD, was excellent, although the calculated ECD may be prone to measurement artifact.

The Effect of Phacoemulsification Energy on the Redox State of Cultured Human Corneal Endothelial Cells

OBJECTIVE To evaluate the effects of phacoemulsification energy on the redox state and mitochondrial distribution of cultured human endothelial cells. METHODS Human corneal endothelial cells from fresh banked human donor tissue not suitable for transplantation were harvested and cultured. Cellular autofluorescence images were obtained using an inverted microscope. The redox fluorometric ratio, which can be related to oxidative stress, was calculated as the net value of fluorescence from the 4,6-diamidino-2-phenylindole channel divided by the net value of fluorescence from the fluorescein isothiocyanate-conjugated channel after subtraction of background. For determining the mitochondrial distribution patterns, the cell area was divided by drawing a line halfway between the nuclear and cell membranes. The average fluorescence in the central area was divided by the average fluorescence in the peripheral area. This ratio was compared. RESULTS Human corneal endothelial cells exposed to increasing phacoemulsification times and increasing ultrasonic energy levels displayed dose-dependent decreases in measured redox ratios. Lower redox ratios in response to phacoemulsification did not associate with decreases in cell size or altered patterns of mitochondrial localization. CONCLUSION Redox fluorometry may serve as a useful indicator for the in vitro study of human corneal endothelial cell physiological response to ultrasonic stressors and potentially other nonoxidative stressors. CLINICAL RELEVANCE Redox fluorometry in combination with human corneal endothelial cell morphometric measurements has potential to serve as an indicator of human corneal endothelial cell injury resulting secondary to ultrasound phacoemulsification.

Assessment of attachment factors for primary cultured human corneal endothelial cells.

Purpose: To assess the ability of various attachment factors to promote attachment of primary cultured human corneal endothelial cells. Materials and Methods: Primary cultured human corneal endothelial cells (HCEC) were incubated for 2 hours in 24-well plates. Wells had been precoated with commercially available cell attachment improvement media (FNC coating mix), human collagen I, human fibronectin, fibronectin/collagen I, or poly-d-lysine. Ratios of cell count before and after rinsing with culture medium and ratios of cells showing morphological signs of spreading to total cells were calculated to measure effectiveness of attachment factors. Results: Incubation of HCEC for 2 hours in wells without precoating of attachment factors led to a rate of 41 ± 16% (mean ± SD) of cells showing signs of spreading. FNC coating mix, collagen I, and fibronectin/collagen I increased significantly the percentage of cells showing morphological features of attachment at 2 hours. Total cell loss was highest with poly-d-lysine and no pretreatment with attachment factor. Without the use of any attachment factor, 67 ± 19% of cells remained after rinsing. The lowest cell loss was observed with FNC coating mix where 108 ± 5% of cells remained after rinsing. Conclusion: Collagen I, collagen I/fibronectin, and FNC coating mix significantly enhance the spreading of human corneal endothelial cells to tissue culture plates after 2 hours. FNC coating mix significantly reduces cell loss due to rinsing. Without the use of any attachment factor, 67% of the cells remained in situ after rinsing.

Evaluation of Irradiated Corneas Using Scatterometry and Light and Electron Microscopy

Objective: To determine if sterilization of donor corneas by irradiation alters optical, histologic, or ultrastructural tissue characteristics. Methods: Blinded assessment of 10 irradiated and 10 nonirradiated donor corneas by a scatterometer used to objectively measure backscattered corneal light. Light and electron microscopy were performed on samples from both groups. Results: The mean scattering of the nonirradiated (control) corneas was significantly lower (0.0060 ± 0.0034, mean ± SD) than the scattering of the irradiated corneas (0.023 ± 0.0078; P = 0.0001). There was no statistical difference between ages, days in storage, or central corneal thickness of the 2 groups. Light microscopic evaluation revealed attenuation of the irradiated corneal epithelium with superficial stromal clefting. Transmission electron microscopy demonstrated similar fibril diameter in the nonirradiated and irradiated corneas. Although the mean interfibrillar distance was similar between the nonirradiated and irradiated groups, the interfibrillar distance in the superficial stroma was increased in the nonirradiated compared with the irradiated groups (15.4 ± 2.4 vs. 11.8 ± 0.98 nm; P ≤ 0.05). Conclusions: Our data quantitatively support increased light scatter after irradiation, which cannot be attributed to increased corneal thickness. Irradiation alters the histology of the epithelium and the ultrastructure of the superficial corneal stroma. Increased light scatter because of epithelial changes would not be expected to impact postoperative visual function. However, increased light scatter because of stromal changes may affect postoperative visual function if used for optical corneal rehabilitation. Further investigation is warranted to determine whether attenuated epithelium or superficial stromal changes are the cause of increased light scatter in the irradiated corneal tissue.

Evaluation of phacoemulsification‐induced oxidative stress and damage of cultured human corneal endothelial cells in different solutions using redox fluorometry microscopy

Acta Ophthalmol. 2010: 88: e323–e327