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Method Article

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

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DOI:

10.3791/63605

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March 1st, 2022

 ,  , 
1Institute of Biotechnology, HiLIFE, University of Helsinki, 2Institute for Neurosciences of Montpellier, Univ Montpellier

In This Article

Summary

This protocol focuses on damaging the ocular surface of zebrafish through abrasion to assess the subsequent wound closure at the cellular level. This approach exploits an ocular burr to partly remove the corneal epithelium and uses scanning electron microscopy to track changes in cell morphology during wound closure.

Abstract

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. Indeed, the eye injuries most frequently encountered clinically are those to the cornea. While corneal wound healing has been extensively studied in small mammals (i.e., mice, rats, and rabbits), corneal physiology studies have neglected other species, including zebrafish, despite zebrafish being a classic research model.

This report describes a method of performing a corneal abrasion on zebrafish. The wound is performed in vivo on anesthetized fish using an ocular burr. This method allows for a reproducible epithelial wound, leaving the rest of the eye intact. After abrasion, wound closure is monitored over the course of 3 h, after which the wound is reepithelialized. By using scanning electron microscopy, followed by image processing, the epithelial cell shape, and apical protrusions can be investigated to study the various steps during corneal epithelial wound closure.

The characteristics of the zebrafish model permit study of the epithelial tissue physiology and the collective behavior of the epithelial cells when the tissue is challenged. Furthermore, the use of a model deprived of the influence of the tear film can produce new answers regarding corneal response to stress. Finally, this model also allows the delineation of the cellular and molecular events involved in any epithelial tissue subjected to a physical wound. This method can be applied to the evaluation of drug effectiveness in preclinical testing.

Introduction

As most of the epithelia are in contact with the external environment, they are prone to physical injury, making them well suited for the study of wound healing processes. Among the well-studied tissues, the cornea is an extremely useful model in the investigation of the cellular and molecular aspects of wound healing. As a transparent external surface, it provides physical protection to the eye and is the first element to focus the light onto the retina. While the structure and cell composition of the retina differ between species1, these elements of the cornea are generally similar in all camera-type eyes, regardless of species.

The cornea is composed of three main layers2. The first and outermost layer is the epithelium, which is constantly renewed to ensure its transparency. The second layer is the stroma, which contains scattered cells, called keratocytes, within a thick layer of strictly organized collagen fibers. The third and innermost layer is the endothelium, which allows nutrient and liquid diffusion from the anterior chamber to the outer layers. The epithelial and stromal cells interact via growth factors and cytokines3. This interaction is highlighted by the rapid apoptosis and subsequent proliferation of keratocytes after epithelial injury4,5. In case of a deeper wound, such as a puncture, keratocytes take an active part in the healing process6.

Being in contact with the external environment, corneal physical injuries are common. Many of them are caused by small foreign objects7, such as sand or dust. The reflex of eye rubbing can lead to extensive epithelial abrasions and corneal remodelling8. According to wound size and depth, these physical injuries are painful and take several days to heal9. The optimal wound healing characteristics of a model facilitate the understanding of the cellular and molecular aspects of wound closure. Furthermore, such models have also proved useful for testing new molecules with the potential to accelerate corneal healing, as previously demonstrated10,11.

The protocol described here aims to use zebrafish as a relevant model to study corneal physical injury. This model is highly convenient for pharmacological screening studies as it allows molecules to be added directly to the tank water and, therefore, to come into contact with a healing cornea. The details provided here will help scientists perform their studies on the zebrafish model. The in vivo injury is performed with a dulled ocular burr. The impact on epithelial cells adjoining or at a distance from it can be analyzed by specifically removing the central corneal epithelium. In recent years, numerous reports focused on such a method on rodent cornea12,13,14,15,16,17; however, to date, only a single report has applied this method to zebrafish18.

Because of its simplicity, the physical wound is useful in delineating the role of epithelial cells in wound closure. Another well-established model of corneal injury is the chemical burn, especially the alkali burn19,20,21. However, such an approach indirectly damages the entire eye surface, including the peripheral cornea and corneal stroma19. Indeed, alkali burns potentially induce corneal ulcers, perforations, epithelial opacification, and swift neovascularization22, and the uncontrollable outcome of alkali burns disqualifies that approach for general wound healing studies. Numerous other methods are also used to investigate corneal wound healing according to the particular focus of the study in question (e.g., complete epithelial debridement23, the combination of chemical and mechanical injury for partial-thickness wound24, excimer laser ablation for wounds extending to the stroma25). The use of an ocular burr restricts the focal point to the epithelial response to the wound and provides a highly reproducible wound.

As with each method of wound infliction, the use of an ocular burr has advantages and disadvantages. The main disadvantage is that the response being mostly epithelial, it does not perfectly reflect the abrasions seen in the clinical setting. However, this method has numerous advantages, including the ease with which it can be set up and performed, its precision, its reproducibility, and the fact that it is noninvasive, making it a method well tolerated by animals.

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Protocol

All experiments were approved by the national animal experiment board.

1. Preparations

  1. Prepare the tricaine stock solution used for anesthesia26 in advance (0.4% stock solution used in this protocol). Use gloves and keep the materials in a fume hood whenever possible.
    1. For 50 mL of a 0.4% solution, weigh 200 mg of tricaine powder into a 50 mL tube. Dissolve the powder in approximately 45 mL of double-distilled water.
    2. Adjust the pH of the tricaine stock solution to 7 with 1 M Tris (pH 8.8, ~1.25 mL). Add the Tris solution to the tricaine stock in aliquots, mix the stock thoroughly after each aliquot, and check the pH after each addition of Tris.
  2. Before the experiment, prepare a 0.02% working solution of tricaine.
    1. Thaw 2 mL of 0.4% stock solution and add to 40 mL of system water (final concentration 0.02%). Place the solution in a small container.
  3. Before the experiment, prepare the recovery water containing analgesic. Use gloves and keep the materials in fume hood whenever possible.
    1. For one liter of recovery water, weigh 2.5 mg of lidocaine hydrochloride powder and dissolve it in fresh system water. Check the pH and adjust to 7 if necessary.
  4. Before the experiment, prepare the fixing solution (2.5% glutaraldehyde in 0.1 M sodium phosphate (Na-PO4) solution at pH 7.4). Use gloves and keep the materials in a fume hood.
    1. For 10 mL of the fixing solution, pipette 5 mL of 0.2 M Na-PO4 into a tube. Add 0.5 mL of 50% glutaraldehyde, and add double-distilled water to obtain the final volume of 10 mL. Protect the solution from light, and keep it on ice or in the fridge prior to use.
      NOTE: If samples must be collected for several hours after wounding, prepare the fixing solution just prior to use.
  5. Prepare the equipment for wounding (Figure 1).
    1. Fill the recovery tanks or smaller containers with system water.
    2. Have the ophthalmic burr ready. Check that the burr tip is clean. If needed, remove cell debris with a moist cotton swab.
    3. Make an incision to the side of a soft sponge, and moisten the sponge with system water. Place the sponge on the base/stage of a dissecting microscope. Ensure enough working space for using the burr and enough illumination from the sides and/or above to see the eye surface properly.

2. Anesthesia

  1. Transfer a fish from the tank to the 0.02% tricaine solution with a net as gently as possible.
  2. Monitor the anesthesia, checking for lack of response to light mechanical stimulus.
    NOTE: For consistent anesthesia, a 2 min exposure to tricaine is used prior to abrasion with adult wild-type AB fish. With fish of other genetic background, a different duration may be needed.

3. Abrasion

  1. Gently place the anesthetized fish with a spoon into the incision on the sponge, head protruding from the sponge surface.
  2. Turn on the burr, and focus the microscope view onto the eye surface.
  3. Carefully approach the eye surface with the burr tip. When touching the eye surface, start moving the burr tip on the eye surface with circular motion. Avoid sudden movement, as it might lead to the eye tilting in the socket and the burr tip to slip.
  4. When the abrasion is done, carefully place the fish in system water containing the analgesic for recovery.
  5. Clean the burr right after use with a moist cotton swab.

4. Collecting samples

  1. At the desired time point, pick the fish up with a net and place it in 0.02% tricaine solution. Keep the animal in the solution until the opercular movement has ceased completely, and the fish does not react to touching.
  2. Place the fish on a Petri dish with a spoon, and hold it with tweezers. Decapitate the fish with dissecting scissors. Avoid making any scratches on the eye surface when handling the sample.
  3. Put the tissue into a sample tube containing 0.1 M Na-PO4.
    1. Rinse the tissue by replacing the 0.1 M Na-PO4 with clean buffer so that no blood remains in the solution.

5. Sample processing for electron microscopy

  1. Fix the tissue in 2.5% glutaraldehyde/0.1 M Na-PO4 (pH 7.4) for ~24 h at +4 °C. Keep the sample on a rotating/shaking sample holder to ensure proper fixation.
  2. Remove the fixing solution and rinse the sample several times with 0.1 M Na-PO4.
  3. Dissect the sample at this point.
    1. Place the sample onto a drop of 0.1 M Na-PO4 on a dissecting plate. If both eyes from the same fish must be imaged, cut the head sample into two with fine dissecting scissors.
    2. Alternatively, collect the eyes only by carefully placing the tips of fine tweezers into the eye socket from the side of the eye, taking extra care not to scratch the eye surface. Then, pull the eye out from the socket.
    3. Transfer the dissected sample into a tube containing 0.1 M Na-PO4. Ensure there is no extra tissue in the sample tube, as it may adhere to the top of the eye during sample processing.
  4. Store the sample in 0.1 M Na-PO4 (maximum one week) at +4 °C.
  5. Process the samples for electron microscopy imaging.
    1. Postfix the samples in 2% osmium tetroxide in 0.1 M Na-PO4 buffer for 1 h at room temperature (RT).
    2. Wash the samples 3 times for 5 minutes each wash in 0.1 M Na-PO4 at RT.
    3. Dehydrate the samples successively in 30%, 50%, and 70% ethanol for 1 h in each solution at RT.
    4. Immerse the samples in 96% ethanol for 2-3 h at RT.
    5. Next, incubate the samples two times in 100% ethanol, first for 1 h and then in fresh 100% ethanol overnight at +4 °C.
    6. Subject the samples to 30 cycles in an automated critical point dryer.
  6. Embed and platinum-coat the samples.
    1. Place an adhesive tab onto a mount. If the sample must be marked on top of the mount, leave a piece of tab cover paper on the tab and write the sample ID on the paper.
    2. Place the mount with the tab on the base of a dissecting microscope.
    3. Gently place the tissue sample on the mount with fine tweezers, cornea facing up.
    4. Coat the specimen with platinum using the appropriate device. After coating, store the samples at room temperature until imaging.

6. Imaging (Figure 2)

  1. Operate the devices as advised in the user's manual and by imaging experts.
  2. Acquire images of the desired magnification, and use 2,000-2,500x images for analysis.
  3. Adjust the brightness and contrast so that there are no overexposed areas in the image, and cell borders and microridges are seen as clearly as possible.
    NOTE: The position and angle of the tissue affect the brightness and contrast settings. They may need to be adjusted from sample to sample and between different regions of the tissue.

7. Measuring cell shape, size, and microridge pattern

  1. Open the TIFF image in Fiji ImageJ 1.5327. Set the scale using the scale bar of the image: create a line equal in size to the scale bar with the Line tool. Select Analyze | Set scale, and type in the known distance. Open the ROI manager from the Analyze | Tools menu.
  2. For cell size and roundness, select Analyze | Set measurements | Shape descriptors. Use the Magnifying glass tool to see the cells under magnification. Select cells with the Polygon tool, and add each selection to the ROI manager. Finally, measure the selected cells, and save the measurement.
  3. Microridge analysis (Figure 3 and Figure 4)
    1. Ensure that the image is in 8-bit format from the Image | Type menu.
    2. Select a cell with the Polygon tool, and clear the background from Edit | Clear outside.
    3. Smoothen the image one to three times by selecting Process | Smooth, and adjust the brightness and contrast from Image | Adjust | Brightness/Contrast so that the microridges stand out as clearly as possible.
    4. Convolve the image from Process | Filters | Convolve, turn into binary from Process | Binary | Make binary, and skeletonize the black-and-white image by selecting Process | Binary | Skeletonize.
    5. Use the Analyze skeleton function in the Analyze | Skeleton menu to measure the microridge parameters and save the values.
      NOTE: In SEM, individual images may differ in brightness and contrast. Thus, the steps in the analysis may need adjustments from image to image.

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Results

This study describes a method using an ophthalmic burr in zebrafish corneal wound healing experiments. The method is modified from previous studies on mice, where the burr was shown to remove the epithelial cell layers efficiently13. The challenges in zebrafish corneal wounding include the relatively small size of the eye, and in the case of time-consuming experiments, the need to maintain a constant water flow through the gills (as described by Xu and colleagues28). The ma...

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Discussion

Corneal physical injuries are the most common cause of ophthalmology patient visits to the hospital. Therefore, it is important to establish relevant models for the study of different aspects of corneal pathophysiology. So far, the mouse is the most commonly used model for the study of corneal wound healing. However, adding eyedrops on murine wounded eyes to validate the impact of specific drugs on corneal wound healing can be difficult. In this respect, the zebrafish model is particularly useful for the pharmacological ...

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

The authors thank Pertti Panula for the access to the Zebrafish unit and Henri Koivula for the guidance and help with the zebrafish experiments. This research was supported by the Academy of Finland, the Jane and Aatos Erkko Foundation, the Finnish Cultural Foundation, and the ATIP-Avenir Program. Imaging was performed at the Electron Microscopy unit and the Light Microscopy Unit, Institute of Biotechnology, supported by HiLIFE and Biocenter Finland.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.1M Na-PO4 (sodium phosphate buffer), pH 7.4in-houseSolution is prepared from 1M sodium phosphate buffer (1M Na2 HPO4 adjusted to pH 7.4 with 1M NaH2 PO4 ).
0.2M Na-PO4 (sodium phosphate buffer), pH 7.4in-houseSolution is prepared from 1M sodium phosphate buffer (1M Na2 HPO4 adjusted to pH 7.4 with 1M NaH2 PO4 ).
0.5mm burr tipsAlger Equipment CompanyBU-5S
1M Tris, pH 8.8in-house
adhesive tabsAgar ScientificG3347N
Algerbrush burr, Complete instrumentAlger Equipment CompanyBR2-5
Cotton swapsHeinz Herenz Hamburg1030128
Dissecting platein-house
Dissecting toolsFine Science Tools
double-distilled waterin-house
Eppedorf tubes, 2mlany provider
Ethyl 3-aminobenzoate methanesulfonate saltSigmaA5040Caution: causes irritation.
Glutaraldehyde, 50% aqueous solution, grade ISigmaG7651Caution: toxic.
Lidocaine hydrochlorideSigmaL5647Caution: toxic.
mountsAgar ScientificG301P
Petri dishThermo Scientific101VR20
pH indicator stripsMacherey-Nagel92110
Plastic spoonsany provider
Plastic tubes, 15 mlGreiner Bio-One188271
Plastic tubes, 50 mlGreiner Bio-One227261
Scanning electron microscopeFEIQuanta 250 FEG
Soft spongeany provider
Sputter coaterQuorum TechnologiesGQ150TS
StereomicroscopeLeica

References

  1. Baden, T., Euler, T., Berens, P. Understanding the retinal basis of vision across species. Nature Reviews.Neuroscience. 21 (1), 5-20 (2020).
  2. Nishida, T., Saika, S., Morishige, N. Cornea and sclera: Anatomy and physiology. Cornea: Fundamentals, diagnosis and management, 4th ed. Manis, M. J., Holland, E. J. , Elsevier. 1-22 (2017).
  3. Wilson, S. E., Liu, J. J., Mohan, R. R. Stromal-epithelial interactions in the cornea. Progress in Retinal and Eye Research. 18 (3), 293-309 (1999).
  4. Wilson, S. E., et al. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Experimental Eye Research. 62 (4), 325-327 (1996).
  5. Zieske, J. D., Guimaraes, S. R., Hutcheon, A. E. Kinetics of keratocyte proliferation in response to epithelial debridement. Experimental Eye Research. 72 (1), 33-39 (2001).
  6. West-Mays, J. A., Dwivedi, D. J. The keratocyte: corneal stromal cell with variable repair phenotypes. The International Journal of Biochemistry & Cell Biology. 38 (10), 1625-1631 (2006).
  7. Ahmed, F., House, R. J., Feldman, B. H. Corneal abrasions and corneal foreign bodies. Primary Care. 42 (3), 363-375 (2015).
  8. Ben-Eli, H., Erdinest, N., Solomon, A. Pathogenesis and complications of chronic eye rubbing in ocular allergy. Current Opinion in Allergy and Clinical Immunology. 19 (5), 526-534 (2019).
  9. Wilson, S. A., Last, A. Management of corneal abrasions. American Family Physician. 70 (1), 123-128 (2004).
  10. Nagata, M., et al. JBP485 promotes corneal epithelial wound healing. Scientific Reports. 5, 14776(2015).
  11. Wang, X., et al. MANF promotes diabetic corneal epithelial wound healing and nerve regeneration by attenuating hyperglycemia-induced endoplasmic reticulum stress. Diabetes. 69 (6), 1264-1278 (2020).
  12. Li, F. J., et al. Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit. Experimental Eye Research. 147, 1-11 (2016).
  13. Kalha, S., Kuony, A., Michon, F. Corneal epithelial abrasion with ocular burr as a model for cornea wound healing. Journal of Visualized Experiments:JoVE. (137), e58071(2018).
  14. Kalha, S., et al. Bmi1+ progenitor cell dynamics in murine cornea during homeostasis and wound healing. Stem Cells. 36 (4), 562-573 (2018).
  15. Park, M., et al. Visualizing the contribution of keratin-14(+) limbal epithelial precursors in corneal wound healing. Stem Cell Reports. 12 (1), 14-28 (2019).
  16. Kuony, A., et al. Ectodysplasin-A signaling is a key integrator in the lacrimal gland-cornea feedback loop. Development. 146 (14), (2019).
  17. Farrelly, O., et al. Two-photon live imaging of single corneal stem cells reveals compartmentalized organization of the limbal niche. Cell Stem Cell. 28 (7), 1233-1247 (2021).
  18. Ikkala, K., Michon, F., Stratoulias, V. Unilateral Zebrafish corneal injury induces bilateral cell plasticity supporting wound closure. Scientific Reports. , (2021).
  19. Ormerod, L. D., Abelson, M. B., Kenyon, K. R. Standard models of corneal injury using alkali-immersed filter discs. Investigative Ophthalmology & Visual Science. 30 (10), 2148-2153 (1989).
  20. Anderson, C., Zhou, Q., Wang, S. An alkali-burn injury model of corneal neovascularization in the mouse. Journal of visualized experiments: JoVE. (86), e51159(2014).
  21. Choi, H., et al. Comprehensive modeling of corneal alkali injury in the rat eye. Current Eye Research. 42 (10), 1348-1357 (2017).
  22. Singh, P., Tyagi, M., Kumar, Y., Gupta, K. K., Sharma, P. D. Ocular chemical injuries and their management. Oman Journal of Ophthalmology. 6 (2), 83-86 (2013).
  23. Pal-Ghosh, S. BALB/c and C57BL6 mouse strains vary in their ability to heal corneal epithelial debridement wounds. Experimental Eye Research. 87 (5), 478-486 (2008).
  24. Chen, J. J., Tseng, S. C. Abnormal corneal epithelial wound healing in partial-thickness removal of limbal epithelium. Investigative Ophthalmology & Visual Science. 32 (8), 2219-2233 (1991).
  25. Xeroudaki, M., Peebo, B., Germundsson, J., Fagerholm, P., Lagali, N. RGTA in corneal wound healing after transepithelial laser ablation in a rabbit model: a randomized, blinded, placebo-controlled study. Acta Ophthalmologica. 94 (7), 685-691 (2016).
  26. Westerfield, M. The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio. , Available from: https://zfinorg/zf_info/zfbook/zfbk.html (2000).
  27. Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9 (7), 676-682 (2012).
  28. Xu, C., Volkery, S., Siekmann, A. F. Intubation-based anesthesia for long-term time-lapse imaging of adult zebrafish. Nature Protocols. 10 (12), 2064-2073 (2015).
  29. Crosson, C. E., Klyce, S. D., Beuerman, R. W. Epithelial wound closure in the rabbit cornea. A biphasic process. Investigative Ophthalmology & Visual Science. 27 (4), 464-473 (1986).
  30. Parlanti, P., et al. Axonal debris accumulates in corneal epithelial cells after intraepithelial corneal nerves are damaged: A focused Ion Beam Scanning Electron Microscopy (FIB-SEM) study. Experimental Eye Research. 194, 107998(2020).
  31. Zhao, X. C., et al. The zebrafish cornea: structure and development. Investigative Ophthalmology & Visual Science. 47 (10), 4341-4348 (2006).
  32. Richardson, R., et al. Re-epithelialization of cutaneous wounds in adult zebrafish combines mechanisms of wound closure in embryonic and adult mammals. Development. 143 (12), 2077-2088 (2016).
  33. van Loon, A. P., Erofeev, I. S., Maryshev, I. V., Goryachev, A. B., Sagasti, A. Cortical contraction drives the 3D patterning of epithelial cell surfaces. The Journal of Cell Biology. 219 (3), (2020).
  34. Vihtelic, T. S., Hyde, D. R. Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina. Journal of Neurobiology. 44 (3), 289-307 (2000).
  35. Poss, K. D., Wilson, L. G., Keating, M. T. Heart regeneration in zebrafish. Science. 298 (5601), 2188-2190 (2002).
  36. Becker, T., Wullimann, M. F., Becker, C. G., Bernhardt, R. R., Schachner, M. Axonal regrowth after spinal cord transection in adult zebrafish. The Journal of Comparative Neurology. 377 (4), 577-595 (1997).
  37. Hu, X., et al. Sirt6 deficiency impairs corneal epithelial wound healing. Aging. 10 (8), 1932-1946 (2018).
  38. Ksander, B. R., et al. ABCB5 is a limbal stem cell gene required for corneal development and repair. Nature. 511 (7509), 353-357 (2014).
  39. Pan, Y. A., et al. Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish. Development. 140 (13), 2835-2846 (2013).

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Zebrafish CorneaCorneal AbrasionScanning Electron MicroscopyEpithelial Cell ShapeMicroridge AnalysisImageJ AnalysisEpithelial Tissue PhysiologyPharmacological Studies
Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis
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