Induction of Retinal Ischemia-Reperfusion Injury in a Mouse Eye Model

Our research focuses on understanding how the eye naturally prevents inflammation. Our research has shown that the myecorn pathway is essential for suppressing inflammation in the eye and promoting cell survival in the stressed retina. This suggests that inactivating the myecorn pathway could be beneficial for treating retinal generative diseases.

This protocol normalizes intraocular pressure before needle withdrawal, reducing trauma to ocular structures and limiting injury to the ischemia-reperfusion component. This refinement minimizes experimental variability caused by physical manipulation, enhancing the accuracy of this model. Our research promotes the unique concept of therapy instead of neutralizing molecules to lessen symptoms in the hope that cells can survive and return to normal.

We focused on changing the behavior of cells involved in the disease. This approach has a strong potential to provide long-term benefits and resistance to recurrence of chronic diseases, since normal cellular activity is induced. Our model has offered a refined technique to study ischemia-reperfusion injury in the mouse eye.

And this may offer a more accurate representation of how intraocular structures would respond to an injury from prolonged intraocular pressure or from a reperfusion injury. To begin, prepare syringes of sterile 0.9%sodium chloride saline solution connected to an infusion line and a stopcock with a 30-gauge needle attached to the end of the line, one for each mouse, and place them 120 centimeters above the bench surface. Then, flush out all air bubbles from the line.

Next, create a bed to stabilize the mouse by cutting a depression of approximately six by three centimeters in a sponge and placing it securely in a styrofoam well or another flat container. After anesthetizing the mouse, dilate the iris with one drop of 1%tropicamide and allow five minutes for dilation. Then, securely place the mouse in the sponge bed under the microscope.

Apply saline solution to the eyes to rinse away any debris or fur from the eye surface. Using non-toothed forceps, gently hold one of the eyes. Then, with the infusion line closed, cannulate the anterior chamber with the needle approximately two millimeters from the limbus.

Ensure the needle pierces the cornea perpendicularly to the peripheral curved cornea surface, then slightly flattened parallel to the plane of the iris. Turn the stopcock to run the infusion line and verify that there is no leakage through the corneal wound. Ensure a gradual distension of the cornea as the intraocular pressure increases.

Using a tonometer, check that the intraocular pressure has elevated to 90 to 100 millimeters of mercury. Secure the infusion line with tape, ensuring the needle does not change position and start leaking. Now, apply an ophthalmic ointment such as bacitracin to the eye to prevent dryness.

Place the mouse away from the microscope under a heating lamp to maintain normal body temperature for 60 minutes. After 60 minutes, return the mouse to the microscope. Lower the saline solution syringe to the mouse's level to normalize the intraocular pressure.

Measure the intraocular pressure with the tonometer to check that it is near normal, around 20 millimeters of mercury. Once normalized, carefully remove the needle, avoiding damage to the lens or iris. Coat the mouse eyes with an ophthalmic antibacterial ointment, such as bacitracin veterinary ophthalmic ointment.

Monitor the mouse on a heated surface for one to two hours until it fully recovers from anesthesia. Once fully recovered, return the mouse to its cage. Retinas on day seven showed greater cell damage and loss compared to day two, indicating more severe retinal degeneration over time following ischemia and reperfusion.

Individual histopathological scores were higher in most layers of the retina on day seven compared to day two, showing increased pathological changes over time. Total histological scores were significantly higher on day seven than day two, indicating a marked progression in retinal damage.