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Mechanical signaling plays a crucial role in maintaining extracellular matrix (ECM) homeostasis in various structures. In this study, we investigated the responses of corneal fibroblasts to cyclic stretching loads using an in vitro cell culture system. Bovine corneal fibroblasts were cultured and subjected to equibiaxial cyclic strain of 15% for 72 h at a frequency of 0.25 Hz, with bovine skin fibroblasts used as a comparison. We explored various cellular behaviors, including morphological changes, cell proliferation, and metabolism in response to mechanical stretching loads. The expression of genes, protein secretion, and enzymatic activity for several major metalloproteinases was also determined through Q-PCR, Western blot, and gel zymography. Additionally, we investigated the involvement of mitogen-activated protein kinases (MAPKs) signaling pathways in the corneal fibroblasts when subjected to mechanical stimuli. Our findings revealed that, compared to skin fibroblasts, corneal fibroblasts were reluctant to morphological changes in response to a prolonged (72 h) and high-amplitude (15% of strain) cyclic stretching load. However, cyclic stretching loads stimulated the upregulation of MMP-2 expression in corneal fibroblasts via the MAPK signaling pathways involving extracellular signal-regulated kinase and p38. Together with a lack of upregulation in type I collagen expression, our results indicate the induction of the ECM degradation process in corneal fibroblasts in response to cyclic stretching. These findings emphasize the mechanoresponsive nature of corneal fibroblasts and shed light on the potential impact of intense mechanical stress on the cornea in both normal and pathological conditions such as keratoconus, providing valuable insights for understanding corneal mechanobiology., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: To generate a single-cell RNA-sequencing (scRNA-seq) map and construct cell-cell communication networks of mouse corneas., Methods: C57BL/6 mouse corneas were dissociated to single cells and subjected to scRNA-seq. Cell populations were clustered and annotated for bioinformatic analysis using the R package "Seurat." Differential expression patterns were validated and spatially mapped with whole-mount immunofluorescence staining. Global intercellular signaling networks were constructed using CellChat., Results: Unbiased clustering of scRNA-seq transcriptomes of 14,732 cells from 40 corneas revealed 17 cell clusters of six major cell types: nine epithelial cell, three keratocyte, two corneal endothelial cell, and one each of immune cell, vascular endothelial cell, and fibroblast clusters. The nine epithelial cell subtypes included quiescent limbal stem cells, transit-amplifying cells, and differentiated cells from corneas and two minor conjunctival epithelial clusters. CellChat analysis provided an atlas of the complex intercellular signaling communications among all cell types., Conclusions: We constructed a complete single-cell transcriptomic map and the complex signaling cross-talk among all cell types of the cornea, which can be used as a foundation atlas for further research on the cornea. This study also deepens the understanding of the cellular heterogeneity and heterotypic cell-cell interaction within corneas.

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Multiphoton microscopy allows long-term direct visualization of cells in live animals due to its low photodamage. When coupled with fluorescence protein targeting and second harmonic generation signals from natural collagen as contrast, multiphoton microscopy enables intravital tracing of cells while providing structural information from the extracellular matrix. Compared with conventional histological analysis, it can bring new insight into the cell dynamics in stem cell research. Here, we demonstrate cell imaging and tracing at a single cell resolution in the cornea, skin, and hair follicles using multiphoton microscopy in transgenic mice of which specific cell populations are tagged with fluorescent proteins.

The purpose of this study is to provide an intravital noninvasive multiphoton microscopic platform for long-term ocular imaging in transgenic fluorescent mice with subcellular resolution. A multiphoton microscopic system with tunable laser output was employed. We designed a mouse holder incorporated with stereotaxic motorized stage for in vivo three-dimensional imaging of ocular surface in 3 transgenic mouse line with fluorescent protein (FP) expression to visualize distinct structures. With our imaging platform and the expression of FPs, we obtained the three-dimensional images across the whole cornea from epithelium to endothelium and in conjunctiva with subcellular resolution in vivo. Specified EGFP expression in corneal epithelium of K5-H2B-EGFP mice helped to identify both corneal and limbal epithelial cells while ubiquitous nuclear FP expression in R26R-GR mice allowed us to visualized nuclei of all cell types. Universal membrane-localized FP in mT/mG mice outlined all cell boundaries, nerve fibers, and capillaries. The simultaneously collected second harmonic generation signals from collagenous stroma provided architectural contrast. Time-lapsed recording enabled monitoring the mitotic activity of corneal epithelial cells and limbal epithelial cells. We developed an intravital multiphoton microscopic stereotaxic imaging platform and showed that, by incorporating FP-expressing transgenic mice, this platform enables in vivo 4-dimensional ophthalmic study at subcellular resolution., (Copyright © 2019. Published by Elsevier Ltd.)

The cornea is a potential route of exposure and drug administration for nanoparticles. In this work, we use noninvasive two-photon microscopic imaging to study the distribution and permeability pathway of CdSe/ZnS core/shell quantum dots (QDs) capped with three different functional groups through the cornea. With no additional staining, the two-photon image clearly discloses that fluorescent QDs penetrate and reside within the interlamellar space of second harmonic generating collagenous stroma when the corneal epithelium barrier is injured. An in vitro cytotoxicity test using bovine corneal stromal cells incubated individually with all three kinds of QDs indicates that the cell viability decreases significantly as the QD concentration and incubation period increased. The results also show that the specific QDs influence corneal stromal cell viability up to a significant magnitude of 50% under a relatively low concentration (5-20 nM) and short exposure period (24-48 h). Furthermore, two-photon imaging shows that QDs can be retained within the cornea up to 26 days in an in vivo mouse model. On the basis of our in vivo and in vitro data, we conclude that QDs can penetrate and be retained within cornea long enough to cause consequential cytotoxicity, under the circumstance in which the corneal epithelium barrier is injured. Since corneal abrasion is quite a common situation in daily life, our work raises public attention to the potential risk of eye exposure to nanoparticles.

The purpose of this study is to demonstrate the application of multiphoton fluorescence and second harmonic generation (SHG) microscopy for the ex-vivo visualization of human corneal morphological alterations due to infectious processes. The structural alterations of both cellular and collagenous components can be respectively demonstrated using fluorescence and SHG imaging. In addition, pathogens with fluorescence may be identified within turbid specimens. Our results show that multiphoton microscopy is effective for identifying structural alterations due to corneal infections without the need of histological processing. With additional developments, multiphoton microscopy has the potential to be developed into an imaging technique effective in the clinical diagnosis and monitoring of corneal infections.

Purpose: To demonstrate the application of multiphoton fluorescence (MF) and second harmonic generation (SHG) microscopy for ex vivo characterization of the structural alterations of human corneas due to keratoconus., Methods: Buttons of keratoconic corneas derived from penetrating keratoplasty were sent for structural analysis with a custom-built multiphoton microscope. Fluorescence detected within the cytoplasm and the SHG signal obtained from collagen were used to demonstrate the morphologic changes in the corneal specimens., Results: The fluorescent epithelial cells around the apical area were elongated and were aligned parallel to the adjacent collagen fibers. Parallel and centripetal distribution patterns of stromal collagen bundles were demonstrated at different depths within the keratoconic corneas., Conclusions: MF and SHG microscopy provides three-dimensional structural analysis of keratoconus ex vivo. It may provide important morphologic information for the investigation of the pathogenesis of keratoconus and may have potential in a clinical setting as an in vivo diagnostic and monitoring system for advancing keratoconus.

Purpose: The purpose of this work was to demonstrate the use of the combined imaging modality of multiphoton autofluorescence and second-harmonic generation (SHG) microscopy in obtaining spectrally resolved morphologic features of the cornea, limbus, conjunctiva, and sclera in whole, ex vivo porcine eyes., Methods: The 780-nm output of a femtosecond, titanium-sapphire laser was used to induce broadband autofluorescence (435-700 nm) and SHG (390 nm) from various regions of the surface of ex vivo porcine eyes. A water-immersion objective was used for convenient imaging of the curved surface of the eye., Results: Multiphoton autofluorescence was useful in identifying cellular structures of the different domains of the ocular surface, and the SHG signal can be used to resolve collagen organization within the cornea stroma and sclera of ex vivo porcine eyes., Conclusions: Multiphoton autofluorescence and SHG microscopy have been demonstrated to be an effective technique for resolving, respectively, the cellular and collagen structures within the ocular surface of ex vivo porcine eyes. SHG imaging resolved the difference in structural orientations between corneal and sclera collagen fibers. Specifically, the corneal collagen is organized in a depth-dependent fashion, whereas the scleral collagen is randomly packed. Because this technique does not require histologic preparation procedures, it has the potential to be applied for in vivo studies with minimal disturbance to the eye.

We characterize the structural changes of porcine corneal structures from 25 to 90 degrees C using second harmonic generation (SHG) microscopy. Our results show that porcine stroma undergoes several distinct stages of structural changes between 25 and 90 degrees C. A decrease in SHG intensity from 30 to 45 degrees C and the existence of SHG intensity peaks at 53, 65, and 77 degrees C correlate to distinct structural alterations of the corneal stroma. At higher temperatures, the SHG intensity decreases and a baseline in SHG signal is reached at 90 degrees C. Our results demonstrate that SHG microscopy is a useful technique for obtaining qualitative and quantitative information of thermally treated corneal fibers without histological or labeling procedures. With additional developments, SHG imaging may be developed into an effective imaging technique for in vivo characterization of cornea structural changes.

Expansion microscopy (ExM) has gained increasing popularity for 3D ultrastructural imaging of cultured cells and tissue slices at nanoscale resolution using conventional microscopes via physical expansion of biological tissues. However, its application to collagen‐abundant thick tissues is still challenging. Herein, a new method, collagen ExM (ColExM), optimized for expanding tissues containing more than 70% collagen, is demonstrated. ColExM succeeds in 4.5‐fold linear expansion with minimal structural distortion of corneal and skin tissues. It is compatible with immunostaining, allowing super‐resolution visualization of 3D neural structures innervating hair follicles, corneas, and pancreatic tumors with high stromal collagen content. The method succeeds in identifying individual mitochondria and previously unrecognized dendritic spinelike structures of corneal nerves. It also enables fine mapping of structural rearrangement of tight junctions and actin cytoskeletons. Therefore, ColExM can facilitate the exploration of 3D nanoscale structures in collagen‐rich tissues. [ABSTRACT FROM AUTHOR]