Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

The overall goal of this procedure is to induce iris vasculature, which allows direct in-vivo visualization of angiogenic processes. This method can help answer key questions in the ophthalmology field, particularly in neovascular diseases. The main advantage of this technique is that it utilizes albino mice to allow for directing vivo visualization of the blood vessels.

Through this method can give insight into ocular neovascular disease. It can also be applied as a general model of angiogenesis, such as testing for new angiogenic therapies. Visual demonstration of this method is critical, as the iris dissection is challenging because of the small size of the mouse eye.

With practice, however, it can be mastered. After anesthetizing a P12.5 mouse and preparing it for the procedure, put it in the lateral recumbancy position under a stereoscope and confirm the mask is well positioned. The eye should fill the field of view.

Clear focus of the eye is imperative. Next, using small tying forceps in the non-dominant hand, carefully protrude the eye by applying downward pressure to the eyelids, dorsal and ventral to the eye. Keeping a firm hold on the eyelid helps.

Before proceeding, photo document the vasculature at 40X. Next, use the stereoscope to locate the corneal limbus. The limbus of albino BALB/c mice can be easily identified as the circular vascular plexus posterior to the cornea.

Now, in the dominant hand, hold a 30 gauge beveled needle and use it to perform a small uveal puncture near the posterior limbal limit of the uvea between the ciliary body and the ora serrata. Make the puncture using only the tip of the needle and no more than half the bevel. Avoid rupturing the lens.

The puncture should be self-sealing but still allow for intra-ocular injection of study substances through the wound. Next, perform a second uveal puncture in the opposite site of the eye. Optimize the punctures to be at the most dorsal and most ventral positions.

Repeat the uveal punctures at the same two positions every four days until P24.5. And prior to repeating the puncture, always check for traumatic cataract. After enucleating the eye and fixing it as described in the text protocol, remove the fixative solution and rinse the eye three times with fresh PBS.

Then, place the eye on a smooth, dry surface with the anterior chamber upwards. Next, insert a beveled 30 gauge needle posterior to the limbus to make an entry point. Then, secure the anterior segment with small tying forceps and use Clayman-Vannas straight scissors to cut around the limbus while rotating the eye.

Using partial cuts can make the process easier. When completed, the posterior segment of the eye should release. Next, position the anterior segment downward to expose the lens.

Remove the lens carefully by grabbing it with small forceps and pulling upwards. Now, position the anterior segment with the cornea facing down and with the cut perpendicular to the field of view. Then, gently grab the tissue posterior to the ciliary body using small tying forceps in the non-dominant hand.

Clayman-Vannas straight scissors to trim just anterior to the ciliary body with the goal of removing the trabecular meshwork and isolating the iris. The iris should then move within the cornea. Next, use small tying forceps to grip the cornea and carefully transfer the anterior eye cup to a 96-well plate containing 200 microliters of PBS.

Maintain the grip on the cornea and continue dissecting the iris by flushing the tissue with PBS. If needed, a 30 gauge needle can be used to help displace the iris. Now, remove the cornea using the small tying forceps.

The isolated iris can then be stored in PBS at four degrees Celsius temporarily until it is experimented upon. Albino BALB/c mice at P12.5 were subjected to uveal punctures repeated every fourth day until P24.5, as described. Due to the transparency of the cornea and the melanin deficiency of BALB/c mice, iris vasculature is readily visible in P16.5 pups and intensifies throughout the duration of the protocol.

At P27.5, mice were euthanized and their irises were carefully dissected. The punctures induced a vascular response from the iris by triggering the wound healing system. Immunohistochemistry was performed against PECAM1.

The resulting stain showed an increase in overall vasculature in irises of punctured eyes compared to controls. After watching this video, you should have a good understanding of how to induce iris angiogenesis and successfully isolate the mouse iris. Following this procedure, other methods like QBCR can be performed in order to answer additional molecular and cellular questions such as testing the efficacy of pro-angiogenic or anti-angiogenic substances.

After its development, this technique has paved the way for researchers to explore neovascular diseases by in-vivo, non-invasive methods using small rodents as a model.