Previous studies show that the photorefractive keratectomy or transepithelial PRK, is one of the best animal model for the study of corneal healing, and also, the changes associated to histopathological debridement of the epithelial cornea. Working with Mongolian gerbils as animal model has several advantages over other rodents. First, the cornea has the same histologic layers as the human cornea.
Second, the eye is anatomically larger than the eye of other rodents, allowing for easier manipulation. All this research was carried out in the facilities of the Center for Research and Development in Immunology and Infectious Diseases of the Universidad Catolica De Cordoba. The first step was to disinfect the working surface of the laminar flow cabinet.
For this purpose, 70%alcohol was used. Surgical instruments to be used. Disposable materials such as needles and syringes, and drugs to anesthetize the rodent, were placed in the work area.
A disposable surgical field should also be placed to contain secretions and rodent hair if necessary. Three six-month old male Mongolian gerbils of similar size and weight, approximately 90 grams were used in the experiment. The procedures to be performed later were performed on the right eyes only.
The cage with the rodent is taken and placed inside the laminar flow cabinet. We proceed to open the box, identify each of the gerbils, and weigh them on the scale. They're harmless but delicate animals.
Disposable gloves should be worn at all times. For it correct handling, the operator should use his unskilled hand to hold it firmly by the tail, and with the skilled hand, gently press down with a thumb and index finger behind the ears to hold the animal and with the little finger, hold the tail. Based on the weight of the animal, the amount of anesthesia needed for each of the gerbils is calculated.
A one milliliter syringe should be filled with ketamine, 50 over 100 milligram per kilogram, and xylazine, two milligram per kilogram. Prior to performing the optical coherence tomography, sterile surgical drapes were placed to protect the equipment from secretions or animal hair. While one of the operators hold the animal, another operator must take the captures.
The eye was photographed microscopically and multiple slices were made of the interior segment of the rodent. The entire cornea, the anterior chamber, the iris, the iridocorneal angle and lens, which protrudes into the anterior chamber can be observed. In addition, central and peripheral corneal thickness measurements were taken showing a central thickness of 160 micrometers decreasing to approximately 110 micrometers in the periphery.
Prior to perform excimer laser PTK, sterile surgical drapes were placed to protect the equipment from secretions or animal hair. Between 60 and 62 micrometers of corneal thickness were ablated at the diameter of three millimeters. The cornea was photographed to evaluate immediate morphologic changes.
In this photograph, it can be seen, the eye eyeball of the gerbil right eye. A, irregular surface of the normal eye eyeball is observed. B, photograph taken five minutes after PTK was performed showing that the corneal surface presents irregularities.
See evidence of the corneal ulcer stained with fluorescein. On the OCT, the right arrow mark the area where detritus has accumulated in the center and the left arrow mark the edge of the corneal ulcer. The method of sacrifice used in this experiment was through the use of a carbon dioxide chamber.
They were sacrificed at different time intervals and enucleation of the normal eyeball and post-surgical eyeballs were performed at 24 and 96 hours post-surgery to observe corneal healing. Each enucleated eyeball was placed in formaldehyde and processed by a pathologist. Multiple sections, measurements and photographs were taken.
Histologically, the corneal thickness consists with that obtained by OCT, and there is a progressive thinning towards the periphery. This is because the layers of epithelium and stromal fibers decrease in the periphery. The Mongolian gerbil cornea has the same layers as the human, stratified anterior epithelium with four to six layers of cells representing 28%of the total thickness of the cornea.
Bowman's layer, stroma representing 66%of the total thickness. This cements membrane and endothelium. At 24 hours postsurgery, the corneal ulcer can be seen where the arrow is marked.
Also observed multiple patches of epithelial achantholysis and isolated dyskeratocis, acute subepithelial inflammatory infiltrate, edema at the stromal level. The changes observed in the operated gerbil at 96 hours after PTK are, presence of increased edema, disaggregation of stromal fibers and cells, and it should be noted that the interior epithelium was already completely regenerated and did not present inflammatory infiltrate. The present study show the positive result of the current animal model in gerbils and confirming it as a new research option in corneal wound healing.
To date, there are no publications that analyze, in detail, the anatomy and histology of the Mongolian gerbil cornea. Therefore, we hope that this animal model will be the basis for future scientific investigations.
