Correlation of Multimodal Clinical Imaging With the Whole-Slide- and Superresolution-Based Immunohistological Structure of the Corneal Limbal Stroma

Abstract

Purpose: To identify structural landmarks in the limbal stroma by anterior segment optical coherence tomography (asOCT), asOCT angiography (asOCTa) and in vivo confocal microscopy (IVCM), and correlate findings with immunofluorescence microscopy (IFM).

Methods: The corneal limbus of healthy individuals was examined by IVCM, asOCT, and asOCTa. IFM was performed in cadaveric cornea samples sectioned in clinical imaging planes, with putative markers for limbal niche cells (CD90, N-cadherin, SSEA-4), blood/lymphatic vessels (CD31), nerves (acetyl-α-tubulin, β-III tubulin), limbal epithelial progenitor cells (p63), and hyaluronic acid (HA).

Results: On asOCT, asOCTa, and IVCM superficial and deep limbal vessels could be identified that terminated in the marginal corneal arcade (MCA). Limbal stroma appeared as hyperreflective area between superficial and deep limbal blood vessels. The same blood vessel patterns were identified by IFM. HA localization was identical to hyperreflective stromal structures. Nerves and putative niche cells localized around limbal vessels and were in close contact with the basal limbal epithelium at the level of the MCA. Limbal epithelium was hyperreflective and thinned in the elderly (>60 years, 122.6 ± 36.6 µm; <60 years, 139.9 ± 34.4 µm; P = 0.025) resulting in less visible palisades of Vogt on asOCT (>60 years, 82.6% visible; <60 years, 100% visible; P = 0.011).

Conclusions: Our study proves that limbal vessel plexuses can serve as landmarks to identify corresponding structures in various clinical imaging modalities. The proximity of blood vessels, niche cells, and nerves, confirmed by IFM, may suggest that limbal vascular damage occurs together with niche cell and neural loss. The IFM correlations provided by this study help to detect healthy limbal structures and aid the diagnosis of diseased corneas.

Figure 1.

AsOCT and asOCTa images of a healthy individual. (a, b) Lower limbal PoV imaged with asOCTa (a) and corresponding infrared image (b). The palisade vessels are faintly visible as shown in the red square in a, magnified in the left corner of the image. The blue arrow shows superficial and the yellow arrow indicates deep vessel. (c) Cross-sectional images at the green lines in a and b highlight flow signals of PoV vessels. In the left corner, blue and yellow arrows show the flow signals of the corresponding superficial and deep PoV vessels of a. The black arrow shows a shadowing structure without flow signal, probably a vessel not detected by asOCTa. (d) The same cross-sectional image as in (c), without flow signals. (e–h) Lower temporal limbal vessels imaged with asOCTa. (e) En face image from the area between the segmentation lines in panels (g) and (h). (f) Infrared image of the same area. In the magnified square in e, the blue arrow shows small vessels of the MCA, and the yellow arrow shows a larger, deep vessel. The flow signals of the same vessels are seen in g. Note that MCA vessels are located in the triangular tapered hyperreflective stroma at the anterior part of PoV. (h) In the same cross-section as in g, without flow signals, blood vessels can be identified as hyporeflective lines or dots with a shadowing effect. (i) Superficial vessels of the temporal area imaged with asOCTa; parallel vessel of the PoV and tiny loops of the MCA can be seen (the MCA is only partially visible due to segmentation error). (j) Deep vessels are rather Y-shaped and form one arcade at the PoV and a less complete episcleral arcade approximately 2 mm behind the limbus. (k) Longitudinal section of a PoV of the upper limbus of a healthy individual imaged by high-resolution asOCT; thick limbal epithelium is seen at the level of PoV. Underneath the thickened epithelium, wedge-shaped tapering hyperreflective stroma can be seen, with small hyporeflective MCA vessels (blue arrow). The hyperreflective stroma is in continuation with the hyperreflective subconjunctiva containing subconjunctival vessels (orange arrows), and subconjunctival and episcleral (yellow arrow) vessels seem to merge at the PoV and continue into the MCA in the wedge-shaped hyperreflective stroma underneath the thickened limbal epithelium. Between the hyperreflective areas surrounding the subconjunctival and episcleral vessels, the hyporeflective Tenon's capsule can be seen. The boundaries of the histological limbus are marked with green lines. co, cornea; conj, conjunctiva; TC, Tenon's capsule; TM, trabecular meshwork; SS, scleral spur.

Figure 2.

OCT, IVCM, and immunohistology sections in corresponding planes of limbal structures. (a–c) High-resolution anterior segment OCT images of a human corneal limbal area in three different directions: longitudinal (a), transversal (b), and en face (c). Drawings in the bottom show the direction of the image plane above them (in histological sections, upper and lower limbus were not distinguished). (d–j) In vivo confocal microscopy images—en face (d) and insets (g)—of a human corneal upper limbus. Corresponding immunofluorescence microscopy images are in directions identical to the asOCT and IVCM images: longitudinal (e, h), transversal (f, i), and en face (g, j). Sections were stained with labeled hyaluronic acid binding protein (HABP) to show the presence of HA (red) which localizes in the same position as the hyperreflective stroma seen in the asOCT and IVCM images (e–g). Hyaluronidase treatment depleted HABP staining (h–j), verifying the specificity of HABP binding. The green signal shows the blood vessels (positive for CD31). The vessels became more prominent after hyaluronidase treatment (h–j). The vessels formed a deep, episcleral plexus (yellow arrows) and a superficial plexus (blue arrows). These vessels can be observed in asOCT and IVCM images, as well (a–d, and insets in g, where yellow arrows indicate deep vessels and blue arrows indicate superficial vessels). HA is more abundant around superficial and deep blood vessels than in the limbal stroma located in between them. The red signal also highlights the finger-like structures of the PoV, in both cross-sectional (f) and en face (g) images. The limbal epithelium is stained with anti-pancytokeratin antibodies and appears blue. Limbal epithelial crypts can be seen on the longitudinal sections (e, h) and in the en face OCT (c) and IVCM (d) views of PoV, and are marked with white asterisk. Scale bar: 500 µm (e, h); 200 µm (f, g, i, j).

Figure 3.

Immunfluorescence microscopy images showing CD31, CD90, and vimentin staining. (a) Immunofluorescence staining of en face section of a human corneal limbal area, showing CD31, vimentin, and CD90 with (left) and without (right) nuclear staining. Inset in right picture is an en face OCT limbal image; the blue arrow points to PoV (blue box), the red arrow points to the place of MCA (red box). (b) Magnified image of the area marked by the blue rectangle in a, at the level of PoV blood vessels. Above the DAPI image, the blue framed panel shows the corresponding IVCM image of PoV. The blue arrow points at a vessel running in a PoV stromal projection. (c) Magnified image of the area marked by the red rectangle in a at the level of the MCA. An abundant network of vimentin⁺ (red) fibroblastic cells is present in the stroma. Some vimentin⁺ cells are also CD90⁺ (blue), especially around blood vessels (CD31⁺, green). Below the DAPI image is shown an IVCM image of MCA in the red frame. The red arrow points from MCA arches seen in IVCM at the same structures in the immunohistology picture. (d) Longitudinal section of the limbal area. The inset shows corresponding OCT images; green, blue, and red arrows point at areas on the immunofluorescence image magnified in e to g. Note that pictures are epithelial side down. (e) Magnified image of the subepithelial region of the ScL, marked with a green rectangle in d. (f) Magnified image of the PoV region marked with a red rectangle in d. (g) Magnified image at the level of MCA, marked with a blue rectangle in d. At the SL and PoV, CD90⁺ (blue) mesenchymal cells are located in the deeper stromal layers, in the vicinity of CD31⁺ (green) blood vessels. At the MCA, CD90⁺ cells are located just below the basal epithelial cells, still in the vicinity of blood vessels that are very close to the basal epithelium in this region. For high-resolution images, overlay, immunofluorescence, and DAPI-only panels are shown. co, cornea; conj, conjunctiva; ep, epithelium; str, stroma. Scale bar: 20 µm (b, c, e, g); 40 µm (f).

Figure 4.

Immunfluorescence microscopy with CD31, SSEA4, and N-cadherin staining. (a) Immunofluorescent staining of a longitudinal human corneal limbal section and a corresponding OCT image. Red and green arrows point at the magnified areas marked with boxes. (b, c) High-resolution Airyscan image of the area marked by the red and green rectangles in a at the level of deep and superficial PoV vessels, respectively. (d) En face section of the limbal area. Next to it the corresponding view of a limbus on en face OCT and infrared (IR) images are shown. Vessels of PoV and some of the MCA are seen on the IR image. Scale bars: 500 µm. (e) High-resolution image at the level of superficial PoV stroma marked with a red rectangle in a. (f) High-resolution image at the level of the deep blood vessels underneath the PoV marked with a green rectangle in a. For high-resolution images, individual immunofluorescence channels and their overlay with DAPI are shown. SSEA4⁺ (red) epithelial cells are seen in the basal and suprabasal layer of limbal epithelium (b, e). In the superficial PoV stroma, some fibroblastic cells, double positive for SSEA4 and N-cadherin (blue), are seen underneath the epithelium. Some SSEA4⁺ cells are also visible in the proximity of deep vessels (c, f). Some basal limbal epithelial cells were N-cadherin⁺. N-cadherin also marked most limbal stromal cells, especially in the superficial stroma and endothelial cells of blood vessels, the latter also being CD31⁺ (green). On the right, the IVCM image of PoV is shown, with the red arrow pointing at superficial and green arrow pointing at deep PoV vessels. co, cornea; Ep, epithelium; str, stroma. Scale bar: 20 µm for immunofluorescence and 50 µm for IVCM.

Figure 5.

Immunfluorescence microscopy with CD31, p63, and N-cadherin staining. (a) Immunofluorescence staining of a transversal human corneal limbal section and corresponding OCT image on the right. Note that images are epithelial side down. (b) High-resolution image of the area marked with a red rectangle in a, involving both superficial and deep blood vessels. Individual immunofluorescence channels, their overlay, and the overlay with DAPI are shown. N-cadherin⁺ (blue) cells are located between CD31⁺ (green) blood vessels and nuclear p63⁺ (red) basal epithelial cells that also show some N-cadherin staining at their basal part (limbal epithelial stem cells). N-cadherin⁺ stromal cells appear to extend their processes towards p63⁺ basal epithelial cells (yellow arrows). Ep, epithelium; str, stroma. Scale bar: 100 µm (a); 20 µm (b).

Figure 6.

Immunfluorescence microscopy with CD31, β-III tubulin, and N-cadherin staining. (a) Immunofluorescence staining of an en face section of the limbus. Corresponding en face OCT, IR, and IVCM images are shown to the right. The green-framed IVCM is from the ScL region, the red-framed IVCM is from the superficial PoV region as indicated by rectangles in the IF image. Green arrows point at PoV, and red arrows point at the scleral limbus, where deep limbal vessels are seen. (b) High-resolution images of the area at the level of the deep PoV vessels of the ScL marked by the red rectangle in a. (c) High-resolution image of a PoV with a central blood vessel marked with a green rectangle in a. Individual immunofluorescence channels and their overlay with DAPI are shown. At the level of the deep vessels of the ScL, large β-III tubulin–positive (red) nerve bundles run circumferentially along deep CD31⁺/N-cadherin⁺ vessels. In the superficial PoV stroma, fine β-III tubulin–positive nerve fibers are seen that enter the basal epithelial layer. The central CD31⁺/N-cadherin⁺ PoV vessel is also visible. N-cadherin staining is also seen in numerous PoV fibroblasts and some basal epithelial cells. Conj, conjunctiva. Scale bar: 500 µm (a); 40 µm (b, c).

Figure 7.

Immunofluorescence microscopy with CD31 and acetyl-α tubulin staining. (a) Immunofluorescence staining of a longitudinal human corneal limbal section and corresponding OCT image (images are epithelial side down). (b) High-resolution images of the area marked by the red rectangle in a. At the region of deep PoV vessels, numerous acetyl-α tubulin–positive neural axons (blue) can be seen that form a network around CD31⁺ (green) vessels. Scale bar: 100 µm (a); 20 µm (b).