This article describes a new animal model developed to study the anatomy and histology of the cornea and its healing processes. This new animal model uses the Mongolian gerbil, which has a cornea with many similarities to the human cornea.
Corneal wound healing studies have been conducted for a long time and have helped to reduce suffering and develop treatments that contribute to improving patients’ eye health. Historically, corneal healing has been studied in rodents such as mice and rats, but these models might not completely mimic human disorders. However, information on other rodents such as Mongolian gerbils (Meriones unguiculatus) is scant in corneal research.
Here, we describe a technique to develop a novel animal model for studying corneal healing after photorefractive keratectomy. Due to the limited literature available on the cornea of M. unguiculatus, we also describe a histological analysis of the normal cornea. These research techniques can also be employed in the study of eye diseases because of the similarity between the corneas of Mongolian gerbils and humans in terms of genetics, anatomy, and physiology.
Some of the most important aspects of corneal wound healing, which are key concerns for anterior segment surgery, are the integrity of the epithelial architecture, the maintenance of the corneal stroma transparency, and, finally, the outcome in terms of the refractive properties of the cornea1.
The cornea is the outermost clear tissue at the front of the eyeball and is, therefore, susceptible to trauma, infections, and burns; the impaired healing of these wounds can compromise visual health2.
At present, several animal models are available to study corneal healing, and some of them are better than others, depending on the species and the type of mechanism to be studied1. There are a few records of previous investigations on the retina of gerbils2. However, so far, there is no published literature on the scarring processes in the cornea of these rodents.
Here, we present Meriones unguiculatus (Mongolian gerbil) as an animal model of wound healing in the cornea. Procedures to elicit corneal healing after photorefractive keratectomy are described, which allow us to study the different types of corneal scarring processes, understand wound healing in terms of the dynamic phases of living tissue, and, finally, plan appropriate future treatments3. Phototherapeutic keratectomy is a highly reproducible technique with the possibility of precisely controlling parameters such as the depth and diameter of the corneal injury4. Moreover, this technique does not require procedures with surgical instruments or chemical solutions (e.g., saline solution, formalin, alcohol, etc.) that may add variables that are specific to the instruments or to the operator performing the procedure5.
Three 6-month-old male gerbils of similar sizes and weights (approximately 90 g) were used for the experiment presented in this article. The procedures were only performed in the right eyes.One gerbil (referred to as gerbil 1 or control) did not undergo phototherapeutic keratectomy and was enucleated to evaluate all the normal ocular structures. Phototherapeutic keratectomy involves the controlled delivery of excimer laser-generated ultraviolet light to the cornea and was developed in order to perform refractive surgery6. It has been used in other rodents, such as mice7. The other two gerbils were subjected to phototherapeutic keratectomy. One of them was enucleated at 24 h (referred to as gerbil 2) and the other one at 96 h after surgery (referred to as gerbil 3).
To perform this experiment, a gerbil selected at random was filmed for each condition to be studied, but this experiment was previously performed with 16 gerbils in total for each condition. For editing reasons, it was decided to use a randomly selected gerbil for each condition (three gerbils in total) as an example.
The main objective of this research is to explore the best animal model available. However, it is important to note that not all species have eye characteristics similar to those of the human eye8. This article describes the methodology used to study the cornea of Meriones unguiculatus and the procedure performed to generate the corneal injury, which allow us to study the healing process.
All research procedures were approved by the "Institutional Commission for the Care and Use of Laboratory Animals" of Universidad Católica de Córdoba and followed the National Research Council Guide for the care and use of laboratory animals. These procedures were also approved by the authorities of the "Facultad de Ciencias de la Salud" of Universidad Católica de Córdoba and "Instituto de la Visión Cerro".
1. Gerbil handling and anesthesia
NOTE: All animals were specific pathogen-free(SFP) male Mongolian gerbils and were kept in the Center for Research and Development in Immunology and Infectious Diseases (CIDIE) facilities (Córdoba, Argentina). They were obtained from Universidad de La Plata (Buenos Aires, Argentina).
2. Optical coherence tomography (OCT) of the cornea
3. Excimer laser phototherapeutic keratectomy (PTK)
4. Awakening of the gerbils after corneal PTK
5. Euthanasia method
6. Eye surgery
7. Anatomo-pathological analysis
In the present study, the entire corneal structure was thoroughly analyzed using histological techniques and complementary studies of the anterior segment, such as optical coherence tomography. The image analysis using optical coherence tomography of the anterior segment structures shows a normal epithelium and stroma (Figure 1), with central and peripheral corneal thicknesses of 160 µm and 106 µm ± 2 µm, respectively. Other publications have also shown that the cornea of other rodents becomes thinner toward the periphery10.
After PTK, the debridement of the corneal epithelium was observed (Figure 2). Macroscopic pictures of the gerbil eye before and after treatment were also taken. After performing PTK, an irregular corneal surface was observed, which was stained by instilling a drop of fluorescein and illuminating it with violet light (showing the epithelial ulcer) (Figure 3).
Regarding the histological analysis, the normal cornea of the untreated gerbil (gerbil 1) showed the same layers as in humans: the stratified anterior epithelium with four to six layers of cells, representing 28% of the total thickness of the cornea, the Bowman's layer, the stroma, representing 66% of the total thickness of the cornea, the Descemet's membrane, and the endothelium (Figure 4 and Figure 5).
The changes observed in gerbil number 2 (24 h after PTK) were an ulcer in the cornea, sphacelation of the adjacent anterior epithelium, multiple spots of epithelial acantholysis and isolated discheratocytes, acute subepithelial inflammatory infiltrate, and edema at the level of the stroma (Figure 6).
The changes observed in gerbil number 3 (96 h after PTK) were the presence of greater edema than in gerbil number 2, disaggregation of the stromal fibers and cells, complete regeneration of the anterior epithelium, and no inflammatory infiltrate (Figure 7).
In summary, histological staining demonstrates the normal wound healing process in the corneal epithelium and superficial stroma, with inflammatory infiltrate and edema.
Figure 1: Representative OCT imaging of the normal cornea. The cornea can be seen in its full size (with a thickness measurement at its apex of 160 µm and thickness measurements of 108 µm and 110 µm in the periphery), and the anterior chamber, the iridocorneal angle, the iris, and the crystalline lens (protruding into the anterior chamber) can also be seen. Please click here to view a larger version of this figure.
Figure 2: Representative OCT imaging of the cornea pre- and post-PTK. (A) Image of the normal cornea. (B) Corneal image 10 min after PTK. The arrow on the left shows the edge of an ulcer with an accumulation of cellular debris, and the arrow on the right also shows debris on the corneal surface typical of the surgery. Please click here to view a larger version of this figure.
Figure 3: Representative macro photograph of the eyeball of the gerbil (right eye). (A) Regular surface of the normal eyeball. (B) Image taken 5 min after PTK was performed, showing irregularities on the corneal surface. (C) Evidence of a corneal ulcer stained with 0.25% fluorescein using a LED light source (violet). Please click here to view a larger version of this figure.
Figure 4: Representative complete histological section (anterior-posterior) of the entire normal cornea of the gerbil (40x) stained with H&E. (A) The peripheral and central fragments are framed. The scale bar is 20 µm. (B–D) The periphery of the cornea shows a thinner epithelium with less stratification and a decreased number of stromal fibers. The figure shows a slightly detached endothelium in the periphery of the cornea, which is due to an artifact of the technique. Consequently, the peripheral corneal thickness is thinner than the central one. (C) The thickness measured is similar to the thickness calculated with the OCT images. Both the epithelium and stroma show a greater thickness at the level of the corneal apex. The scale bar is 40 µm. Please click here to view a larger version of this figure.
Figure 5: Representative normal cornea of the Mongolian gerbil stained with H&E (gerbil number 1). The five layers of the cornea and the intact epithelium are observed. The scale bar is 100 µm. Please click here to view a larger version of this figure.
Figure 6: Representative cornea 24 h after excimer laser phototherapeutic keratectomy (PTK) (stained with H&E). The arrow shows the edge of the corneal ulcer (gerbil number 2). The scale bar is 100 µm. Please click here to view a larger version of this figure.
Figure 7: Representative cornea 96 h after excimer laser phototherapeutic keratectomy (PTK). Stained with H&E; gerbil number 3. The regenerated epithelium and stromal edema are observed in this figure. The scale bar is 100 µm. Please click here to view a larger version of this figure.
The physiology of corneal wound healing is a balance between tissue regeneration and the maintenance of homeostasis. Excessive wound healing may lead to fibrosis and scarring, which ultimately may result in the loss of organ function. With the rapid evolution of corneal surgical procedures, the importance of understanding corneal wound healing and the physiological and pathological events involved cannot be overestimated11.
Multiple research works claim that gerbils have many sensory characteristics that make them a favorable species for vision studies, including mainly diurnal behavior12 and superior and more acute vision compared to mice or rats13. Their retinal structure is more analogous to that of humans14. For this reason, they have been used as an animal model for the development of retinal parasitic infections15, therapeutic drugs, gene delivery, and to study of retinal physiology. In addition, recent published genetic analyses have shown that most of the identified gerbil genes (81%) are shared between mice and humans16. Moreover, studies have documented the genetic similarities between gerbils and both mice and humans, identifying important similarities and differences across species17. Therefore, we chose the current animal model of gerbils to study the normal corneal structures and their pathophysiological processes associated with PTK scarring.
Several researchers argue that PTK is an ideal model to study corneal scarring because it allows the study of apoptotic processes, keratocyte vitality, cell migration, and local tissue inflammation, among other aspects18.
The importance of this work relates not only to the study of corneal scarring and wound healing but also to the proposal of a new animal model with the scientific potential for the results to be extrapolated to other previously published models.
This animal model, due to its similarity and resemblance to the behavior of the human eye, allows for the reproduction of the same protocol with different variants and sets a precedent for the development of other models, such as models of infectious keratitis and corneal neovascularization, among others.
However, this work and this animal model have some limitations. First, the gerbil is not a widespread animal model such as mice, rats, or rabbits. For this reason, there may not be as many reagents as desired. Secondly, the available literature on ophthalmology in gerbils is also very limited.
The authors have nothing to disclose.
We would like to thank engineer Rodrigo de la Fuente for his invaluable help and technical support. We also thank María Eugenia Corbela for the narration and Priscilla Hazrún for the edition of the figures. Hugo Luján allowed us to use the facilities at the Center for Research and Development in Immunology and Infectious Diseases (CIDIE).
Anesthesia | Tododrogas | ||
Eppendorf tubes | Tododrogas | ||
Excimer Laser | Technolas | 2022445 | |
Fluorescein | Poen | ||
Forceps | Ofcor | 3339 | |
Formaldehyde | Tododrogas | ||
Gloves | Tododrogas | ||
Ketamine | Sigma-Aldrich | ||
Optical coherence tomography | Optovue | 659007 | |
Proparacaine | Poen | ||
Scisors | Ofcor | 3336 | |
Sterile drapes | Soporte hospitalario | ||
Sterile gauzes | Soporte hospitalario | ||
Syringes and needles | Tododrogas | ||
Xylazine | Sigma-Aldrich |