July 2009
Volume 50, Issue 7
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Cornea  |   July 2009
Detection of Subepithelial Fibrosis Associated with Corneal Stromal Edema by Second Harmonic Generation Imaging Microscopy
Author Affiliations
  • Naoyuki Morishige
    From the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan; the
  • Naoyuki Yamada
    From the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan; the
  • Shinichiro Teranishi
    From the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan; the
  • Tai-ichiro Chikama
    Department of Ocular Pathophysiology, Yamaguchi University School of Medicine, Yamaguchi, Japan; and the
  • Teruo Nishida
    From the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan; the
  • Atsushi Takahara
    Institute for Materials Chemistry and Engineering, Kyushu University, Fukuoka, Fukuoka, Japan.
Investigative Ophthalmology & Visual Science July 2009, Vol.50, 3145-3150. doi:10.1167/iovs.08-3309
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      Naoyuki Morishige, Naoyuki Yamada, Shinichiro Teranishi, Tai-ichiro Chikama, Teruo Nishida, Atsushi Takahara; Detection of Subepithelial Fibrosis Associated with Corneal Stromal Edema by Second Harmonic Generation Imaging Microscopy. Invest. Ophthalmol. Vis. Sci. 2009;50(7):3145-3150. doi: 10.1167/iovs.08-3309.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

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-mm2 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 μm from the surface of Bowman’s 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 Bowman’s layer in both normal and edematous corneas. Abnormal SHG signals at the level of Bowman’s layer were observed in edematous corneas; three-dimensional images revealed that these signals were actually localized above Bowman’s 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.

Corneal transparency is dependent on an ordered structure of collagen in the corneal stroma and on an intact multilayered structure of the corneal epithelium and functioning corneal endothelial cells. 1 Stromal edema is associated with various corneal diseases, including bullous keratopathy, graft failure after keratoplasty, and Fuchs’ dystrophy, and it results primarily from dysfunction of corneal endothelial cells. The well-aligned structure of collagen fibrils in the stroma is lost with the development of edema. Stromal edema is apparent clinically as a thickening of the stroma. Structural analysis has demonstrated that the anterior stroma of human edematous corneas is not swollen, 2 indicating that the structure of the anterior stroma is stable. Histopathologic observations of corneas affected by diseases such as bullous keratopathy have revealed the accumulation of extracellular matrix proteins, including collagen, 3 tenascin, 3 fibronectin, 3 and fibrillin-1, 4 in the anterior stroma or of extracellular matrix below the epithelium, referred to as subepithelial fibrosis. 5 Such changes in corneal structure may contribute to the development of corneal opacity associated with stromal edema. 
Irradiation with a femtosecond laser gives rise to second harmonic signals from oriented noncentrosymmetric proteins such as collagen, myosin, or tubulin present in the living body. Collagen fibrils are aligned uniformly in the corneal stroma and are therefore thought to be responsible for second harmonic generation (SHG) from the cornea. SHG imaging has thus allowed visualization of collagen organization in the cornea. 6 7 8 9 Such imaging has demonstrated that collagen lamellae are interwoven in the anterior stroma of the normal human cornea 10 11 and that the structure of stromal collagen is altered in corneas affected by keratoconus. 11 12 SHG signals have also allowed visualization of the structural changes of stromal collagen induced by excimer laser photoablation 13 or femtosecond laser photodisruption. 14 15 Furthermore, SHG images can be processed to generate three-dimensional (3D) reconstructions of corneal structure. 
To examine the histopathologic changes of the cornea associated with stromal edema, we investigated the structure of corneal collagen lamellae by SHG imaging microscopy in tissue from individuals with bullous keratopathy, graft failure after penetrating keratoplasty, or Fuchs’ dystrophy as well as in normal donor corneas. In addition, we examined changes in the shape of cells in the stroma associated with edema by fluorescence microscopy of the actin cytoskeleton stained with phalloidin. 
Methods
Tissue Specimens
The study was approved by the Institutional Review Board of Yamaguchi University Hospital and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all subjects. 
Corneal buttons were obtained at the time of penetrating keratoplasty from 31 individuals with stromal edema. Of the 31 corneas sampled, 14 were affected by bullous keratopathy secondary to cataract surgery, glaucoma surgery, or laser iridotomy, 13 were affected by graft failure after penetrating keratoplasty, and 4 were affected by Fuchs’ dystrophy. The duration of stromal edema in each instance was determined from clinical charts (Table 1)
We also collected the anterior segment of the corneal stroma left over from eight donor corneas after DSAEK (Descemet’s stripping automated endothelial keratoplasty), as control tissue (Table 1) . After measurement of corneal thickness under 70 mm Hg of intraocular pressure, the anterior part of the donor cornea with a thickness of ∼350 μm was removed with a microkeratome. The eight corneal flaps were collected immediately after creation of the DSAEK graft. 
Preparation of Tissue
All corneal buttons were transferred to 4% paraformaldehyde immediately after their collection. The tissue was fixed overnight at 4°C, after which smaller (∼3-mm2) blocks were dissected from the central region, washed with phosphate-buffered saline (PBS), and stained overnight at 4°C with a red fluorescent nucleic acid stain (Syto 59; Molecular Probes, Eugene, OR) and Alexa Fluor 488–conjugated phalloidin (Molecular Probes) in PBS to identify nuclei (DNA) and the actin cytoskeleton, respectively. The tissue blocks were then mounted on glass coverslips with 50% glycerol in PBS and imaged. 
SHG Imaging Microscopy
Specimens were examined by inverted microscope (Axiovert 200; Carl Zeiss Meditec, GmbH, Jena, Germany, equipped with a 40× [numerical aperture, 1.2] water-immersion objective lens, and having a working distance of 190 μm). Two-photon second harmonic signals from collagen were generated with a mode-locked titanium:sapphire laser (Maitai; Spectra-Physics Lasers Division, Mountain View, CA). The optimal wavelength for the generation of second harmonic signals from human corneal collagen has been found to be 800 nm. 10 Forwardscatter signals or transmitted signals that passed through the tissue were collected with the use of a condenser lens (numerical aperture, 0.55) and a narrow bandpass filter (400/50) positioned in front of the transmission light detector. Backscatter signals were collected by the microscope objective and detected (LSM 510 META detector; Carl Zeiss Meditec, GmbH) over wavelengths from 377 to 430 nm. With the multitrack mode of the detector, we obtained sequential, en face second harmonic and single-photon fluorescence signals from the same optical slice. All samples were scanned with a 1-μm step size in the z-axis to generate 3D data sets extending from the surface of Bowman’s layer to a depth of 150 μm into the anterior stroma. The fluorescence signals from Alexa Fluor 488–phalloidin and fluorescent red nucleic acid stain (Syto 59; Molecular Probes) were excited by the 488- and 633-nm laser lines of the argon and red helium-neon lasers, respectively, and were collected by bandpass filters of 500 to 550 nm and of 650 to 710 nm, respectively. Twelve-bit 512 × 512 images were recorded. The 3D data sets were reconstructed with the use of image software (LSM Image Examiner; Carl Zeiss Meditec, GmbH). A minimum of three 3D data sets was collected from different randomly selected regions of each corneal block. 
Statistical Analysis
Data were analyzed by Fisher’s exact test. P < 0.05 was considered statistically significant. 
Results
SHG signals were obtained from all normal corneas and corneas with stromal edema. The SHG forwardscatter signals from the anterior stroma (∼10 μm below Bowman’s layer) of normal corneas were visualized as short, narrow bundles of linear structures in random orientations (Fig. 1A) . The SHG backscatter signals from the same optical plane had a nonuniform appearance (Fig. 1C) . In the case of edematous corneas, the SHG forwardscatter signals were visualized as short, narrow bundles of linear structures similar to those of the normal cornea (Fig. 1B) . In contrast, the SHG backscatter signals from the same optical plane of corneas with stromal edema appeared more uniform than those observed with the normal cornea (Fig. 1D) . The number of regions characterized by a weak SHG backscatter signal was reduced in edematous corneas (Fig. 1C)compared with that in normal corneas (Figs. 1D)
En face SHG images at the level of Bowman’s layer in normal corneas showed a dotlike pattern of weak forwardscatter signals and a uniform appearance of backscatter signals (Fig. 2A) . Reconstructed 10-μm cross-sectional images derived from stacked SHG images revealed an amorphous structure with a thickness of ∼10 μm corresponding to Bowman’s layer (Fig. 2B) . Projection images derived from all stacked SHG images of each normal cornea showed that the interwoven collagen lamellae in the anterior stroma were adherent to Bowman’s layer (Fig. 2C) , consistent with our previous observations. 10 11 For some corneas with stromal edema, en face images of SHG forward- and backscatter signals revealed an abnormal structure at the level of Bowman’s layer (Fig. 2D) . The SHG forwardscatter signals were not oriented randomly but were aligned with this structure. Reconstructed 10-μm cross-sectional images showed that the abnormal structure revealed by SHG forward- and backscatter signals was actually located immediately above Bowman’s layer (Fig. 2E)and was therefore indicative of subepithelial fibrosis. The projection images derived from all SHG images demonstrated that the abnormal structure was present along the entire surface of Bowman’s layer (Fig. 2F) . The interwoven structure of SHG forward signals appeared to be maintained in the edematous corneas (Fig. 2F)
Projection images of the SHG forwardscatter signal of edematous corneas obtained at various times after the onset of stromal edema are shown in Figure 3 . The image of a cornea affected by stromal edema for 12 months showed the typical interwoven structure of collagen lamellae at the anterior stroma, but abnormal SHG signals at the level of Bowman’s layer were not apparent (Fig. 3A) . The images of corneas affected by stromal edema for 22 (Fig. 3B)or 27 (Fig. 3C)months also showed the interwoven collagen lamellae; however, the abnormal accumulation of collagen fibers was also observed above a thinned Bowman’s layer. The relationship between the duration of clinical stromal edema and the development of subepithelial fibrosis is plotted in Figure 3D . Subepithelial fibrosis was detected only in patients affected by stromal edema for at least 12 months. The mean ± SD duration of stromal edema in patients without subepithelial fibrosis and in those with this condition was 14.8 ± 9.9 and 34.8 ± 19.8 months, respectively. Furthermore, classification of patients into those affected by stromal edema for ≤12 or >12 months revealed that the prevalence of subepithelial fibrosis was significantly greater in the latter group (Table 2)
The shape of cells in the anterior stroma was evaluated by laser confocal microscopy of corneas stained with Alexa Fluor 488–phalloidin and nucleic acid stain (Syto 59; Molecular Probes) to visualize the actin cytoskeleton and nuclei (DNA), respectively. In normal corneas, stromal cells exhibited a stellate or dendritic morphology typical of keratocytes (Fig. 4A) . Whereas cells in the stroma of some corneas affected by edema also manifested typical keratocyte morphologies (Fig. 4B) , those in other edematous corneas appeared more spread out and exhibited a more pronounced actin cytoskeleton (Fig. 4C) , indicative of transdifferentiation into fibroblast-like cells. Examination of the relationship between the presence of such fibroblastic cells and the duration of stromal edema revealed that such cells were detected only in corneas affected by edema for at least 13 months (Fig. 4D) . The mean ± SD duration of stromal edema in patients without fibroblastic cells and in those with them was 19.0 ± 16.4 and 35.8 ± 17.7 months, respectively. Moreover, classification of patients into those affected by stromal edema for ≤12 or >12 months revealed that the prevalence of a fibroblastic morphology for stromal cells was significantly greater in the latter group (Table 3)
Finally, we compared the occurrence of subepithelial fibrosis or fibroblastic transdifferentiation of stromal cells among patients with different conditions responsible for stromal edema (Fig. 5) . These characteristics seemed to develop earlier in patients with bullous keratopathy than in those with graft failure. 
Discussion
With the use of SHG imaging microscopy, we have shown that interwoven collagen lamellae are maintained in the anterior stroma of corneas with stromal edema. We also detected the presence of subepithelial fibrosis in corneas affected by stromal edema with this imaging technique. Furthermore, our data indicate that the duration of stromal edema—in particular a duration of 12 months or longer—is associated with secondary changes in the anterior segment of the cornea. Our findings thus suggest that stromal edema may be a progressive disease associated with time-dependent histopathologic changes in the anterior cornea. 
The presence of subepithelial fibrosis in the edematous cornea was visualized by SHG imaging microscopy as an irregular pattern of forward- and backscatter signals at the level of Bowman’s layer. Immunofluorescence staining has revealed subepithelial fibrosis as the accumulation of several extracellular matrix proteins, including several types of collagen, in corneas with stromal edema. 3 The SHG signal is generated by oriented noncentrosymmetric structures, with that corresponding to subepithelial fibrosis in the edematous cornea being derived from collagen. The development of subepithelial fibrosis was associated with a duration of stromal edema of at least 12 months, indicating that histopathologic changes in the edematous cornea may be progressive, as previously suggested. 3  
Our observations indicate that the interwoven structure of collagen lamellae at the anterior stroma is preserved during stromal edema. Changes in this structure have been proposed to affect the shape of the anterior cornea in keratoconus. 11 The preservation of collagen structure at the anterior stroma is thus consistent with the clinical observations that stromal edema does not affect the anterior shape of the cornea. The anterior segment of the corneal stroma has also been found not to be swollen in individuals with stromal edema. 2  
In addition to subepithelial fibrosis, we detected cells with a fibroblast-like morphology in the stroma of corneas affected by edema for >12 months. During the repair of corneal injury, corneal fibroblasts produce extracellular matrix 16 and matrix metalloproteinases 17 18 as well as downregulate the expression of crystallins, 19 resulting in corneal stromal opacity as well as stromal cell opacity. 20 21 Fibroblasts expressing α-smooth muscle actin (myofibroblasts) were not previously observed in corneas with bullous keratopathy or graft failure, 22 suggesting that keratocytes do not transdifferentiate into myofibroblasts in corneal edematous diseases. Stromal edema may thus result in the transformation of keratocytes into fibroblast-like cells rather than into myofibroblasts, with any associated corneal opacity thus being much milder than that associated with corneal scarring diseases such as postinfectious or postinjury corneal leukoma. 22 23  
Endothelial keratoplasty such as DSEK (Descemet’s stripping endothelial keratoplasty) or DSAEK (Descemet’s stripping automated endothelial keratoplasty) has been performed as an alternative to penetrating keratoplasty in individuals with bullous keratopathy or Fuchs’ dystrophy, with the number of such procedures being on the rise. 24 One concern with endothelial keratoplasty is the timing of the surgery. Our results showing the onset of pathologic changes at around 12 months after the onset of stromal edema suggest that such surgeries should be performed before this time. 
SHG imaging at the level of Bowman’s layer revealed subepithelial fibrosis in the edematous cornea. SHG imaging microscopy generates en face images of the cornea, and both slice images and 3D projection images can be reconstructed from stacked en face images. SHG backscatter signals from Bowman’s layer are detected as amorphous images, whereas SHG forwardscatter signals from Bowman’s layer are weak. 10 Bowman’s layer consists of types I and IV collagen, but electron microscopy has shown that collagen of Bowman’s layer does not have an oriented structure, 25 26 27 28 explaining the weakness of the SHG forwardscatter signals. The condition of Bowman’s layer has been found to affect the structure of collagen lamellae in the anterior stroma as visualized by SHG forwardscatter signals. 11 Analysis of the 3D structure of Bowman’s layer by SHG imaging microscopy may provide insights into the pathologic course of various corneal diseases. 
We observed the 3D structure of collagen lamellae in normal and edematous corneas by analysis of maximal projection images of the SHG forward signals. The SHG microscopic system applied in the present study allowed us to obtain 230-μm2 images in continuous 1-μm steps, yielding a data set including cross-sectional information with a thickness of 230 μm. Analysis of such thick samples is more sensitive for the detection of pathologic changes compared with normal sectioning. Analysis with thin sections may thus fail to detect pathologic changes. Whereas a 10-μm slice image reconstructed from SHG signals revealed subepithelial fibrosis in the edematous cornea (Fig. 2E) , the interwoven structure of anterior collagen lamellae was more readily apparent in projection images (Figs. 2C 2F) . Reconstruction of 3D images obtained by SHG imaging microscopy is thus a sensitive means of analysis of corneal structure. 
The number of regions with a weak SHG backscatter signal was reduced in corneas with stromal edema, giving rise to a “flat” backscatter pattern, compared with that in the normal cornea. The areas of the corneal stroma giving rise to a strong SHG forwardscatter signal have been found not to give rise to a strong backscatter signal. 10 The SHG backscatter signal intensity differs for different tissues, 29 suggesting that the SHG backscatter pattern may depend on the source protein for SHG. The “flat” backscatter pattern of the edematous cornea may thus indicate the accumulation of abnormal collagen around the aligned collagen lamellae. This pattern was observed in all subjects with bullous keratopathy, graft failure, or Fuchs’ dystrophy in the present study, suggesting that abnormal (nonoriented) collagen accumulates during the early phase of stromal edema. The SHG backscatter signal would be detectable if the imaging technique were to be applied to living eyes. Further studies are required to understand the biological meaning of the SHG backscatter signal for the clinical application of SHG imaging to examine collagen organization directly in patients. 
 
Table 1.
 
Characteristics of the Study Subjects
Table 1.
 
Characteristics of the Study Subjects
Subjects n Age (y) Duration of Edema (mo)
Mean ± SD Range Mean ± SD Range
Stromal edema BK 14 69.3 ± 9.4 50–89 24.1 ± 16.2 5–60
GF 13 72.2 ± 12.6 45–91 24.3 ± 31.9 4–65
FD 4 76.0 ± 12.8 60–89 26.3 ± 25.2 12–64
Total 31 71.3 ± 11.5 45–91 24.5 ± 18.3 4–65
Controls 8 71.4 ± 2.2 68–74
Figure 1.
 
SHG images of the anterior segment of the stroma in normal and edematous corneas. (A, B) SHG forwardscatter images of the anterior region (∼10 μm below Bowman’s layer) of a normal cornea and a cornea affected by stromal edema, respectively. Short, narrow bundles of linear structures in random orientations are apparent in both corneas. (C, D) SHG backscatter images from the same optical planes of the normal and edematous corneas shown in (A) and (B), respectively. The normal cornea (C) shows more regions with a weak SHG backscatter signal (arrows) than does the edematous cornea (D). Scale bar, 50 μm.
Figure 1.
 
SHG images of the anterior segment of the stroma in normal and edematous corneas. (A, B) SHG forwardscatter images of the anterior region (∼10 μm below Bowman’s layer) of a normal cornea and a cornea affected by stromal edema, respectively. Short, narrow bundles of linear structures in random orientations are apparent in both corneas. (C, D) SHG backscatter images from the same optical planes of the normal and edematous corneas shown in (A) and (B), respectively. The normal cornea (C) shows more regions with a weak SHG backscatter signal (arrows) than does the edematous cornea (D). Scale bar, 50 μm.
Figure 2.
 
SHG images of Bowman’s layer. (A, D) En face SHG images at the level of Bowman’s layer in normal and edematous corneas, respectively. Forward- and backscatter signals are shown in cyan and magenta, respectively. (B, E) Reconstructed 10-μm slices of the same normal and edematous corneas, respectively. ( Image not available ) Bowman’s layer; (arrow) abnormal accumulation of SHG signals above Bowman’s layer in the cornea affected by stromal edema. (C, F) Reconstructed 3D images derived from all stacked SHG forwardscatter images of the same normal and edematous corneas, respectively. Scale bar, 50 μm.
Figure 2.
 
SHG images of Bowman’s layer. (A, D) En face SHG images at the level of Bowman’s layer in normal and edematous corneas, respectively. Forward- and backscatter signals are shown in cyan and magenta, respectively. (B, E) Reconstructed 10-μm slices of the same normal and edematous corneas, respectively. ( Image not available ) Bowman’s layer; (arrow) abnormal accumulation of SHG signals above Bowman’s layer in the cornea affected by stromal edema. (C, F) Reconstructed 3D images derived from all stacked SHG forwardscatter images of the same normal and edematous corneas, respectively. Scale bar, 50 μm.
Figure 3.
 
Relationship between the duration of stromal edema and the development of subepithelial fibrosis in the cornea. (AC) Projection images derived from stacked images of the SHG forwardscatter signal for corneas affected by stromal edema for 12, 22, or 27 months, respectively. Subepithelial fibrosis (arrowheads) above a thinned Bowman’s layer ( Image not available ) is apparent in (B) and (C) and preservation of the interwoven collagen lamellae (arrow) in the anterior stroma is shown in (C). (D) Plot of corneas with (filled symbols) or without (open symbols) subepithelial fibrosis versus the duration of stromal edema. Scale bar, 50 μm.
Figure 3.
 
Relationship between the duration of stromal edema and the development of subepithelial fibrosis in the cornea. (AC) Projection images derived from stacked images of the SHG forwardscatter signal for corneas affected by stromal edema for 12, 22, or 27 months, respectively. Subepithelial fibrosis (arrowheads) above a thinned Bowman’s layer ( Image not available ) is apparent in (B) and (C) and preservation of the interwoven collagen lamellae (arrow) in the anterior stroma is shown in (C). (D) Plot of corneas with (filled symbols) or without (open symbols) subepithelial fibrosis versus the duration of stromal edema. Scale bar, 50 μm.
Table 2.
 
Relationship between the Duration of Stromal Edema and the Development of Subepithelial Fibrosis
Table 2.
 
Relationship between the Duration of Stromal Edema and the Development of Subepithelial Fibrosis
Duration of Edema (months) Subepithelial Fibrosis Total
+
≤12 1 11 12
>12 14 5 19
Total 15 16 31
Figure 4.
 
Cytoskeletal and nuclear staining of the cornea. (AC) A normal cornea (A) and corneas affected by stromal edema for 12 (B) or 27 (C) months were stained with Alexa Fluor 488-phalloidin and a red fluorescent nucleic acid stain (Syto 59; Molecular Probes, Eugene, OR). Cells in the stroma manifested keratocyte-like (A, B) or fibroblast-like (C) morphologies. (D) Plot of corneas with fibroblastic (filled symbols) or keratocytic (open symbols) morphologies for stromal cells versus the duration of stromal edema. Scale bar, 50 μm.
Figure 4.
 
Cytoskeletal and nuclear staining of the cornea. (AC) A normal cornea (A) and corneas affected by stromal edema for 12 (B) or 27 (C) months were stained with Alexa Fluor 488-phalloidin and a red fluorescent nucleic acid stain (Syto 59; Molecular Probes, Eugene, OR). Cells in the stroma manifested keratocyte-like (A, B) or fibroblast-like (C) morphologies. (D) Plot of corneas with fibroblastic (filled symbols) or keratocytic (open symbols) morphologies for stromal cells versus the duration of stromal edema. Scale bar, 50 μm.
Table 3.
 
Relationship between the Duration of Stromal Edema and the Detection of Fibroblastic Cells in the Stroma
Table 3.
 
Relationship between the Duration of Stromal Edema and the Detection of Fibroblastic Cells in the Stroma
Duration of Edema (months) Fibroblastic Cells Total
+
≤12 0 12 12
>12 10 9 19
Total 10 21 31
Figure 5.
 
Relationship between the duration of stromal edema and the occurrence of subepithelial fibrosis (A) or fibroblastic transdifferentiation of stromal cells (B) for corneas affected by bullous keratopathy (BK), graft failure (GF), or Fuchs’ dystrophy (FD). Filled and open symbols indicate patients positive or negative, respectively, for subepithelial fibrosis or fibroblastic transdifferentiation.
Figure 5.
 
Relationship between the duration of stromal edema and the occurrence of subepithelial fibrosis (A) or fibroblastic transdifferentiation of stromal cells (B) for corneas affected by bullous keratopathy (BK), graft failure (GF), or Fuchs’ dystrophy (FD). Filled and open symbols indicate patients positive or negative, respectively, for subepithelial fibrosis or fibroblastic transdifferentiation.
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Figure 1.
 
SHG images of the anterior segment of the stroma in normal and edematous corneas. (A, B) SHG forwardscatter images of the anterior region (∼10 μm below Bowman’s layer) of a normal cornea and a cornea affected by stromal edema, respectively. Short, narrow bundles of linear structures in random orientations are apparent in both corneas. (C, D) SHG backscatter images from the same optical planes of the normal and edematous corneas shown in (A) and (B), respectively. The normal cornea (C) shows more regions with a weak SHG backscatter signal (arrows) than does the edematous cornea (D). Scale bar, 50 μm.
Figure 1.
 
SHG images of the anterior segment of the stroma in normal and edematous corneas. (A, B) SHG forwardscatter images of the anterior region (∼10 μm below Bowman’s layer) of a normal cornea and a cornea affected by stromal edema, respectively. Short, narrow bundles of linear structures in random orientations are apparent in both corneas. (C, D) SHG backscatter images from the same optical planes of the normal and edematous corneas shown in (A) and (B), respectively. The normal cornea (C) shows more regions with a weak SHG backscatter signal (arrows) than does the edematous cornea (D). Scale bar, 50 μm.
Figure 2.
 
SHG images of Bowman’s layer. (A, D) En face SHG images at the level of Bowman’s layer in normal and edematous corneas, respectively. Forward- and backscatter signals are shown in cyan and magenta, respectively. (B, E) Reconstructed 10-μm slices of the same normal and edematous corneas, respectively. ( Image not available ) Bowman’s layer; (arrow) abnormal accumulation of SHG signals above Bowman’s layer in the cornea affected by stromal edema. (C, F) Reconstructed 3D images derived from all stacked SHG forwardscatter images of the same normal and edematous corneas, respectively. Scale bar, 50 μm.
Figure 2.
 
SHG images of Bowman’s layer. (A, D) En face SHG images at the level of Bowman’s layer in normal and edematous corneas, respectively. Forward- and backscatter signals are shown in cyan and magenta, respectively. (B, E) Reconstructed 10-μm slices of the same normal and edematous corneas, respectively. ( Image not available ) Bowman’s layer; (arrow) abnormal accumulation of SHG signals above Bowman’s layer in the cornea affected by stromal edema. (C, F) Reconstructed 3D images derived from all stacked SHG forwardscatter images of the same normal and edematous corneas, respectively. Scale bar, 50 μm.
Figure 3.
 
Relationship between the duration of stromal edema and the development of subepithelial fibrosis in the cornea. (AC) Projection images derived from stacked images of the SHG forwardscatter signal for corneas affected by stromal edema for 12, 22, or 27 months, respectively. Subepithelial fibrosis (arrowheads) above a thinned Bowman’s layer ( Image not available ) is apparent in (B) and (C) and preservation of the interwoven collagen lamellae (arrow) in the anterior stroma is shown in (C). (D) Plot of corneas with (filled symbols) or without (open symbols) subepithelial fibrosis versus the duration of stromal edema. Scale bar, 50 μm.
Figure 3.
 
Relationship between the duration of stromal edema and the development of subepithelial fibrosis in the cornea. (AC) Projection images derived from stacked images of the SHG forwardscatter signal for corneas affected by stromal edema for 12, 22, or 27 months, respectively. Subepithelial fibrosis (arrowheads) above a thinned Bowman’s layer ( Image not available ) is apparent in (B) and (C) and preservation of the interwoven collagen lamellae (arrow) in the anterior stroma is shown in (C). (D) Plot of corneas with (filled symbols) or without (open symbols) subepithelial fibrosis versus the duration of stromal edema. Scale bar, 50 μm.
Figure 4.
 
Cytoskeletal and nuclear staining of the cornea. (AC) A normal cornea (A) and corneas affected by stromal edema for 12 (B) or 27 (C) months were stained with Alexa Fluor 488-phalloidin and a red fluorescent nucleic acid stain (Syto 59; Molecular Probes, Eugene, OR). Cells in the stroma manifested keratocyte-like (A, B) or fibroblast-like (C) morphologies. (D) Plot of corneas with fibroblastic (filled symbols) or keratocytic (open symbols) morphologies for stromal cells versus the duration of stromal edema. Scale bar, 50 μm.
Figure 4.
 
Cytoskeletal and nuclear staining of the cornea. (AC) A normal cornea (A) and corneas affected by stromal edema for 12 (B) or 27 (C) months were stained with Alexa Fluor 488-phalloidin and a red fluorescent nucleic acid stain (Syto 59; Molecular Probes, Eugene, OR). Cells in the stroma manifested keratocyte-like (A, B) or fibroblast-like (C) morphologies. (D) Plot of corneas with fibroblastic (filled symbols) or keratocytic (open symbols) morphologies for stromal cells versus the duration of stromal edema. Scale bar, 50 μm.
Figure 5.
 
Relationship between the duration of stromal edema and the occurrence of subepithelial fibrosis (A) or fibroblastic transdifferentiation of stromal cells (B) for corneas affected by bullous keratopathy (BK), graft failure (GF), or Fuchs’ dystrophy (FD). Filled and open symbols indicate patients positive or negative, respectively, for subepithelial fibrosis or fibroblastic transdifferentiation.
Figure 5.
 
Relationship between the duration of stromal edema and the occurrence of subepithelial fibrosis (A) or fibroblastic transdifferentiation of stromal cells (B) for corneas affected by bullous keratopathy (BK), graft failure (GF), or Fuchs’ dystrophy (FD). Filled and open symbols indicate patients positive or negative, respectively, for subepithelial fibrosis or fibroblastic transdifferentiation.
Table 1.
 
Characteristics of the Study Subjects
Table 1.
 
Characteristics of the Study Subjects
Subjects n Age (y) Duration of Edema (mo)
Mean ± SD Range Mean ± SD Range
Stromal edema BK 14 69.3 ± 9.4 50–89 24.1 ± 16.2 5–60
GF 13 72.2 ± 12.6 45–91 24.3 ± 31.9 4–65
FD 4 76.0 ± 12.8 60–89 26.3 ± 25.2 12–64
Total 31 71.3 ± 11.5 45–91 24.5 ± 18.3 4–65
Controls 8 71.4 ± 2.2 68–74
Table 2.
 
Relationship between the Duration of Stromal Edema and the Development of Subepithelial Fibrosis
Table 2.
 
Relationship between the Duration of Stromal Edema and the Development of Subepithelial Fibrosis
Duration of Edema (months) Subepithelial Fibrosis Total
+
≤12 1 11 12
>12 14 5 19
Total 15 16 31
Table 3.
 
Relationship between the Duration of Stromal Edema and the Detection of Fibroblastic Cells in the Stroma
Table 3.
 
Relationship between the Duration of Stromal Edema and the Detection of Fibroblastic Cells in the Stroma
Duration of Edema (months) Fibroblastic Cells Total
+
≤12 0 12 12
>12 10 9 19
Total 10 21 31
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