Mongolian Gerbils as an Animal Model of Wound Healing

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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).

1. House the gerbils socially in polysulfone cages bedded with corncob bedding. Provide food and filtered tap water in water bottles ad libitum. Ensure the room temperature range is 18 °C to 24 °C and a 12:12 h light-dark cycle is used.
2. Weigh each gerbil separately, and identify each one to avoid confusion. Make a mark with an indelible ink marker on the base of the gerbil's tail. Use tweezers to hold the gerbil's ear and make a mark using the indelible marker on the rodent's ear. If a laboratory and a large biotherium are available, assign a unique cage for each rodent with the corresponding identification.
3. Disinfect the laminar flow hood with 70% ethanol solution. Place all the surgical and disposable instruments including needles, syringes, and racks in the work area inside the hood. Place a disposable surgical foam in the hood as well.
4. Use a small open plastic container to keep the rodent on the precision balance to facilitate measurement.
5. Use three 6-month-old male Mongolian gerbils of similar sizes and weights (~80 g) for this experiment. One at a time, place each cage with the rodent inside the laminar flow hood. Open the cages, identify each of the gerbils, and weigh them on the scale.
   NOTE: Gerbils are harmless but delicate animals. Wear disposable gloves when handling the gerbils.
6. Grasp the gerbil with the non-dominant hand to hold it firmly by the tail. Use the dominant hand, with the thumb and index finger behind the ears, to hold the animal with the ventral region facing upward. Use the little finger to hold the tail.
7. Fill a syringe with a 30 G needle with 1 mL of ketamine and xylazine. Administer the anesthesia intraperitoneally into the rodent (50-100 mg/kg ketamine and 2 mg/kg xylazine)⁹ using the dominant hand. The duration of the effect will be approximately 20-50 min (variations may occur).
8. To ensure the gerbil is fully anesthetized, check with a toe pinch, tail pinch, and corneal reflection, etc., prior to making incisions in the cornea.
   ​NOTE: Perform all the procedures in the right eye only.

2. Optical coherence tomography (OCT) of the cornea

1. Place sterile surgical drapes to protect the equipment from secretions or animal hair.
2. Ensure one of the operators holds the animal while another operator takes the images. The operator should rest his hands on the equipment while holding the gerbil so that the gerbil's eye is as stable and still as possible to be studied. Rest the hand holding the gerbil on the chin rest..
3. Start the software that controls the OCT, and press Take Image and then Save Desired Image. Perform multiple sagittal and coronal slices of the cornea. Image the eye under the OCT, and make multiple slices to view the anterior segment of the rodent cornea.
   NOTE: If the obtained image is not sharp and the eye has moved slightly, repeat the procedure several times to obtain enough images.
4. Using the OCT software, perform pachymetric measurements of the central and peripheral regions. In the main screen of the software, press Take Image, and then press the Save Desired Image button.
5. Perform measurements on the normal or control eye and immediately after phototherapeutic keratectomy on the other rodent eyes.

3. Excimer laser phototherapeutic keratectomy (PTK)

1. Place sterile surgical drapes on the excimer laser device to protect the equipment from secretions or animal hair.
2. Instill a drop of topical proparacaine hydrochloride (0.5%) in the eye to be treated 5 min before the surgical procedure.
3. Use the non-dominant hand to hold the gerbil firmly. With the dominant hand, open the animal's eyelids so that the images can be captured properly. To be able to focus and obtain a sharp image, ensure that the hands of the person holding the gerbil rest on the head of the equipment. Place the hands holding the animal where a patient would place their neck.
4. Perform PTK ablation on the right eye. Use the following parameters: an ablation between 60 µm and 62 µm thick, an optical zone of 3 mm, a duration of 4 s, and a total of 1,867 pulses.
   NOTE: PTK is performed only on gerbil 2 and gerbil 3. In this step, the second operator prepares and activates the laser to ablate the corneal tissue.
5. Immediately after the procedure, take photographs and perform OCT analysis to record and document surface changes in the treated eyes.
6. Once the procedure is completed, place the rodent back in the cage, monitor the vital signs (heart rate: 360 beats per minute; rectal temperature: 37-38.5 °C; respiratory rate: 90 breaths per minute), and let the animal recover from the anesthesia.

4. Awakening of the gerbils after corneal PTK

1. Administer buprenorphine (0.1mg/kg to 0.05mg/kg) and atipamezole (0.1-1 mg/kg) via intraperitoneal injections.
2. Place each gerbil in its respective home cage, and monitor the vital signs for normal awakening (the normal body temperature is 37-39 °C).
3. Apply an erythromycin ointment to keep the surface clean and prevent infection. Perform this procedure twice per day.
4. Administer buprenorphine (every 6-12 h) subcutaneously (0.01-0.05 mL) for analgesia and eye ointment on two consecutive days after PTK.

5. Euthanasia method

1. Perform euthanasia in the home cage whenever possible.
2. Introduce compressed carbon dioxide (CO₂) gas into the home cage. A filling rate of 30%-70% of the chamber volume per minute with CO₂ added to the existing air in the home cage is adequate to achieve a mixture that meets the objective (for a 10 L volume chamber, use a flow rate of 3-7 L/min). Use cervical dislocation (as a secondary method of euthanasia) to ensure the death of the rodent.
3. At 24 h and 96 h after surgery for gerbil 2 and gerbil 3, respectively, remove the animal from the home cage to perform the enucleation of the eyeball (both the normal eyeball and the one undergoing surgery) to observe corneal healing.
4. Place the animal on the operating table, and check for the absence of a heartbeat for approximately 1 min.

6. Eye surgery

1. Remove the upper and lower eyelids to gain access to the eyeball. Use surgical forceps and scissors to remove the eyelids. The size of the working area is so small and delicate that removing the eyelids allows the eyeball to be enucleated without damaging it.
2. To enucleate the eyeball, make an incision in the external canthus, and guide the scissors in a posterior direction. Repeat this procedure from the internal canthus by separating the eyeball from the orbit.
3. Section the optic nerve at the back of the eyeball. It should be clarified that the posterior orbital plexus usually generates slight bleeding when performing this technique, which makes the work difficult.
4. Introduce the eyeball into a microcentrifuge tube with sterile saline solution for 30 s to 1 min to wash out any residual blood.
5. Place the eyeball in a microcentrifuge tube containing 10% formaldehyde for subsequent anatomo-pathological analysis as described below. Take multiple images and photographs.

7. Anatomo-pathological analysis

1. Embed the whole eye in 10% buffered formalin for 6-24 h.
2. Cut the tissue using a microtome. Ensure that the cut tissue has a thickness of 3 mm.
3. Soak the tissue in 96% alcohol for 30-90 min, and repeat this procedure twice.
4. Place the tissue in isopropyl alcohol for 30-90 min, and repeat this procedure twice.
5. Put the tissue in xylene or xylene substitute for 1-3 h.
6. Embed the tissue in liquid kerosene for 1 h minimum.
7. Use a block to place the tissue and embed it in liquid paraffin. Allow it to solidify (place it in a cold place), and cut it.
8. Prepare the microtome for sectioning according to the manufacturer's instructions.
9. Subsequently, use stains such as hematoxylin and eosin.
10. Obtain pictures with the camera added to the microscope.

Representative Results

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.

Discussion

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.

Materials

Name	Company	Catalog Number	Comments
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