This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.
The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.
Epithelial layers of numerous organs are exposed to injuries. However, they also contain the ability to compensate for tissue loss through wound healing. The cornea offers an excellent model to study wound healing. It forms the external surface of the eye and provides a protective layer for the sensitive ocular machinery. Thus, cornea functions as a physical barrier to pathogens and water loss. It is composed of three layers; epithelium, stroma and endothelium. The epithelium of the cornea makes up the outermost layer of the cornea. Epithelial cells maintain the barrier function of the cornea by adhering strictly to each other through tight junctions1,2,3. An acellular corneal basement membrane, the Bowman's membrane, separates the epithelium from the extensive stroma, which contains refractory keratocytes. Under the stroma, endothelial cells channel nutrients, water, and oxygen to the upper layer.
Corneal abrasions are very common in the clinic4. Injuries to the cornea are diverse, but are largely caused by small particles such as dust or sand, scratches, or other foreign objects. The protocol described here aims at reproducing a clinically relevant type of corneal epithelial abrasion. In doing so, this protocol provides a controllable and seminal method for clinicians and corneal scientists to implement in their own studies. We have performed an in vivo injury repair assay on the murine cornea by abrading the tissue with a dulled ocular burr, the Algerbrush II. Here, we target the abrasion only to the central corneal epithelium and leave the other parts of the organ without damage. Thus, the protocol is ideal to study corneal epithelial cell dynamics or the basement membrane during re-epithelialization, cell migration, proliferation and differentiation in vivo5. Recently, this model was used to analyze progenitor cell dynamics in the murine cornea as well as to unveil the capacity of the differentiated corneal epithelial cells in re-establishing the corneal stem cell niche after injury6,7. Following abrasion, the cornea returns to its normal transparency and tensile strength. Interestingly, an in vitro study indicated that re-epithelialization occurs without increased cell proliferation8. This protocol describes the timeline of uninterrupted healing in the murine cornea. The method is thus applicable to test the effect of drugs on healing patterns and speed.
The cornea has been extensively used for wound healing studies. However, many studies have relied on other models of injury. A well-established model of corneal injury is the alkaline burn that is performed by applying sodium hydroxide (NaOH) with or without filter paper on the corneal surface9. Alkaline exposure results in a large and diffuse injury that affects not only the corneal epithelium, but also the conjunctiva and stroma9,10. Strong alkaline solutions have been shown to induce corneal ulcers, opacification, and neovascularization9. Inflammatory cells invade the stroma typically within 6 h and remain there until 24 h11. Thus, alkaline injury is an advisable method in studies related to stromal activation. Another type of chemical injury can be inflicted by applying dimethyl sulfoxide (DMSO) on the cornea9,10. Other commonly used injury models include incisional wounds that penetrate through the stroma and keratectomy wounds, which are limited to the upper portion of the stroma14,15. These methods are also useful to answer questions regarding stromal wound healing. Different injury models have their own advantages and disadvantages. Abrasion, or debridement, of the corneal epithelium was first developed using dulled scalpels or blades on ex vivo corneas16. This method has later been used in vivo on mouse, rat, and rabbit17,18,19,20,21,22. Using the ocular burr (Figure 1), we remove only a selected region of the epithelium, leaving the rest of the epithelium unaffected. This way, it is possible to precisely target the epithelial removal to different parts of the cornea. In addition, the abrasion size can be assessed with fluorescein staining. Furthermore, here we follow abrasion closure during the healing period.
This method poses several advantages, i) including precise location of abrasion site, which is not possible with chemical injury, ii) the abrasion is quick to perform, and iii) it is non-invasive. Herein, we describe the method using the outbred NMRI mouse as a model, however this could be applied to the vast array of mouse genetic models, as well as to the rat and rabbit, which are common models used to study human corneal disruption.
All experiments are approved by the national animal experiment board.
1. Preparations
2. Corneal Abrasion
CAUTION: Use protective wear (gloves, lab coat) when handling mice. Weigh the mouse to estimate the volume of medication to administer.
3. Imaging the Abrasion
4. Waking up after Corneal Abrasion
5. Imaging during Wound Healing
6. Cornea Collection and Paraffin Embedding
7. Paraffin Sections of the Cornea
This protocol describes a model to inflict an abrasion injury to the mouse cornea and suggests how to follow and visualize the healing process after abrasion. Recently, we employed this method to study the role of corneal epithelial progenitor cells during wound healing6. The use of established tools is the key to a successful abrasion experiment. We, and others, have used the Algerbrush II ocular burr (Figure 1) to perform the abrasions6,7,24. This tool has a dulled burr tip of 0.5 mm in size that brushes rather than drills or shears the corneal epithelium away. Thus, this tool is the recommended choice for an abrasion injury on the cornea.
A central part of this model is that the affected region can be effortlessly visualized at desired intervals. In Figure 3A, we present the use of fluorescein staining in combination with a Cobalt blue light source (Figure 1) to display the abrasion site. In this experiment, we performed a large wound in the central cornea and left the peripheral cornea untouched (Figure 3A). Furthermore, we abraded only the left eye of each mouse and kept the right, bilateral eye as a non-abraded control. The bilateral eye samples remained negative for fluorescein signal, which suggests that they do not undergo any cell loss during the experiment and thus function well as control samples. However, green signal in the borders of the eye displayed the fluorescein solution that accumulated in the junction between the eye and the eyelid. The abrasion was largest at 0 h, immediately following the injury (Figure 3A). It was markedly smaller after 18 h and, in this case, located close to the upper border of the eye. However, the epithelium closes the exposed region at an equal speed from all sides of the abrasion. Notably, at 72 h post-wounding, no green signal was visible. This indicates that the corneal epithelium was fully re-epithelialized by 72 h after the abrasion.
With this method, we aimed at focusing the abrasion only on the corneal epithelium, so that deeper layers of the cornea remained intact. The removal of the corneal epithelium was evident from histological sections in Figure 3B. At 0 h, the edge of the abrasion is shown as a narrow, acellular ledge that is continuous from a regular, 4-5 cell layers thick corneal epithelium. The limbus, peripheral border of the corneal epithelium, presents an example region that was not affected by the abrasion during the entire experimental timeline (72 h). In addition, histological sections indicated that the deeper layers of the cornea, the stroma and the endothelium, were not harmed by the ocular burr. These two tissues appeared similar in abraded and bilateral eye samples. At 18 h post-injury, the healing process is active and re-epithelialization is ongoing. This was suggested by the appearance of a leading edge. The leading edge contains only 1-2 epithelial cell layers and it covers the exposed region before re-stratification can occur25. In line with the results on Figure 3A, the surface was fully re-epithelialized by 72 h, when the migrating fronts had covered the wound and all the epithelial layers were again present. Histological sections from the bilateral eye confirmed (Figure 3A) that the control eyes remained intact during the observation period.
Lastly, we provide evidence that the abrasion model is restricted to the corneal epithelium and does not invoke a stromal response to injury, such as neovascularization. Figure 4 shows the murine cornea at 18 h after abrasion. Based on macroscopic view, this time point did not display a stromal response, thus indicating that our protocol does not disrupt the deeper corneal layers. In comparison, an alkaline injury with 0.75 mol/L NaOH immediately induced neovascularization in the stroma. Given the rapid neovascularization in alkaline injury, the time points in our method provide evidence to rule out the possibility of response in the stroma.
Figure 1: Essential tools for corneal epithelial abrasion. On the left is a vibrating ocular burr. The Cobalt blue pen light in the right is used to bring the fluorescein stain visible. Please click here to view a larger version of this figure.
Figure 2: Imaging the corneal abrasion. (A) Tools for imaging are an adjustable camera arm with clamp (left), the SLR camera (center) and a table lamp with flexible arm and clamp (right). The Cobalt blue pen light is tied to the dome of the lamp with two cable ties. (B) A general view of the imaging setup; the distance of each attachment tool is marked in the image in cm. Both clamps are 6 cm away from the heat plate and the mouse is placed 10 cm away from the heat plate edge. (C) A closer look at the abrasion imaging with proposed distance and position to the mouse eye in cm. Please click here to view a larger version of this figure.
Figure 3: Detection and localization of the corneal abrasion. (A) Mouse eyes at 0, 18, and 72 h after abrasion. Fluorescein signal marks the abraded region in bright green, whereas all other regions in the epithelium remain dark. Bilateral eyes serve as controls. Dashed line marks the borders of the wound and the white spot in each eye is the reflection from the camera. (B) Histologically sectioned and hematoxylin and eosin stained samples of the mouse eyes at 0, 18, and 72 h after abrasion. Limbus is the border that surrounds the corneal epithelium from all sides. Asterix marks the edge of the abrasion. Scale bar is 100 µm. Please click here to view a larger version of this figure.
Figure 4: Corneal abrasion does not activate a stromal response. Alkaline exposure with 0.75 mol/L NaOH followed by irrigation with 0.9% NaCl produces corneal neovascularization (eye collected immediately after treatment). Abrasion with the ocular burr shows no neovascularization even after 18 h. Abrasion site is marked with a dashed line. Scale bar 1 mm. Please click here to view a larger version of this figure.
Wounding methods are popular tools to study different aspects of corneal homeostasis and pathologies. The abrasion model offers a well-controlled method to address relevant problems in ophthalmology. However, certain critical points in the protocol are worth emphasizing. Notably, the details outlined regarding the veterinary medicine, wound healing timeline, and outcome are optimized for use with outbred NMRI and ICR stocks, but may vary among strains of mice26. With this protocol, the experimental animals will remain anesthetized for approximately 20 minutes. This gives a short but sufficient window of time to perform the abrasion. However, when using the ocular burr, making the abrasion itself is a very quick operation and should be possible to perform in this time frame.
Easy and accessible visualization is one of the main advantages of the abrasion model. Combined with molecular biology methods, this opens possibilities to study the epithelial cells in great detail. Abraded corneas can be processed for histological or immunological staining's and proteins or nucleic acids can be collected and examined further. Pajoohesh-Ganji et al. showed that gene expression patterns in the corneal epithelial cells change upon corneal insult with a dulled blade27. To ensure controlled sampling, we recommend that images of the abrasions are taken immediately after the operation and sample collections follow exactly the planned timelines.
This protocol has uses beyond examination of corneal epithelialization. For example, abrasion of the corneal limbus, the stem cell niche of the cornea, may be used to study stem cell dynamics7. We showed that the non-abraded region of the epithelium remained normal during the healing process, however some authors claim that the basement membrane and the underlying stromal keratocytes are affected by the ocular burr24,28. The removal of the basement membrane can be estimated with specific basement membrane staining. Furthermore, repeated abrasions over a longer timeline could reveal interesting healing patterns. In this protocol, we did not observe the architecture of the epithelium over a long-term chase. Both dulled scalpel and ocular burr injuries have shown an increased incidence to corneal erosions after one to several weeks after injury24,29,30. This should be kept in mind when using the abrasion model for long-term studies. However, studies focusing on corneal erosions might find this protocol useful.
Other wounding models are described at length by Stepp et al. in a review5. Together, this protocol and the existing approaches provide versatile options to examine both fundamental biological questions as well as practical clinical problems.
The authors have nothing to disclose.
We would like to thank Kaisa Ikkala for her invaluable technical assistance and insightful help when actualizing this method as well as later on during implementation to our central research questions. We would also like to thank the Laboratory Animal Center and Anna Meller for her help with planning the guidelines of the veterinary work.
NMRI mouse | Envigo | 275 | |
0.9% NaCl | use sterile | ||
Medetomidine | Vetmedic | Vnr087896 | Market name: Cepetor Vet |
Ketamine | Intervet | Vnr511485 | Market name: Ketaminol Vet |
Buprenorfin | Invidior | 3015248 | Market name: Temgesic |
Atipamezol | Orion Pharma | Vnr471953 | Market name: Antisedan Vet |
Carprofen | Norbrook | Vnr027579 | Market name: Norocarp Vet |
1% fucidin acid eye ointment | Dechra | Vnr080899 | Market name: Isathal |
Fluorescein salt | Sigma-Aldrich | F6377 | |
Phosphate-buffered saline solution | PBS | ||
Algerbrush ii ocular burr (0.5 mm tip) | Algerbrush | 6.39768E+11 | |
Cobalt Blue pen light | SP Services | DE/003 | |
Hot plate | Kunz Instruments | 2007-0217 | |
Digital SLR camera | Nikon | D80 | |
Adjustable camera arm and clamp | Neewer | 10086132 | Height 28 cm |
Table lamp with a flexible arm and a clamp | Prisma | ||
Soft wipe | KimtechScience | 7552 | |
CO2 chamber | |||
Dissection toolset | Fine Science Tools | ||
Syringes | Beckton Dickinson | 303172 | |
26G needles | Beckton Dickinson | 303800 | |
2 mL Eppendorf tube | Sarstedt | 689 | |
Tissue casette | Sakura Finetech | 4118F | |
Tissue processing machine ASP200S | Leica | ||
Xylene | VWR | UN1307 | |
Paraffin wax | Millipore | K95523361 | |
Tissue embedding mold 32 x 25 x 6 mm | Sakura Finetech | 4123 | |
Microtome | Microm | HM355 | |
Water bath for sectioning | Orthex | 60591 | |
Water bath for sectioning | Leica | HI1210 | |
Microtome blade | Feather | S35 | |
Glass slide | Th.Geyer GmbH & Co. | 7,695,019 | |
Ultrapure water | Millipore | MPGP04001 | MilliQ |
Paraformaldehyde | Sigma-Aldrich | 158127 | PFA |