Live whole-eye, ex vivo imaging and laser-induced micro injury of the corneal basal epithelium and visualization of resident macrophage responses
Bibliographic record
Abstract
Abstract Purpose In this paper, we describe a novel live-imaging approach to visualize the short-term response of the mouse cornea to basal epithelial cell damage. Laser scanning confocal microscopy was used to induce precisely-defined, localized corneal basal epithelial cell damage and the live macrophage response to this damage was visualized and analyzed. Methods Lipophilic fluorescent dyes, SGC5 or FM 4-64, were injected into the anterior chamber of enucleated eyes and imaged live in whole-mount using confocal laser scanning microscopy. Laser-induced damage was performed by focusing onto a defined region of the corneal basal epithelium for a brief period using a high laser power setting and then returning to low laser power for imaging. Eyes from CX3CR1 +/GFP mice were used to observe macrophage responses to laser damage in real-time. Results SGC5 or FM 4-64 dyes injected into the anterior chamber readily enter the cornea and are taken up by the stromal layer and labeled the outer membranes of corneal epithelial cells and remained stable when visualized using low laser power. Subjecting a defined region of the basal epithelium to high laser power for 1 minute or longer led to a rapid internalization of dye in the exposed basal epithelium cells and overall increase in cellular fluorescence. This change in fluorescence was also accompanied by cell swelling and contraction. Cellular internalization of the non-lipophilic, dye Alexa 647 hydrazide, indicated that membranes were compromised indicating that exposure to high power laser stimulation causes cellular damage to the basal epithelium. Visualization of corneal resident macrophages close to the site of laser-induced damage showed that within minutes, projecting macrophage filopodia extended towards the damaged region at a rate of 0.75µm/min for roughly 40 minutes. Conclusion We have developed a novel approach to image the live cornea and its response to damage. Laser-scanning confocal microscopy can be utilized to induce localized damage to mouse corneal basal epithelium and elicit a macrophage morphological response. This approach represents a useful tool for studying corneal wound healing and cellular responses to damage using live whole-mount imaging.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".