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.

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 becom...

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 ha...

Materials

List of materials used in this article

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