Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The first step is to assemble the device from a blood collection set. Cut a 25 and threequarter gauge needle from this eight centimeter length of sterile tubing. With a lure lock, you can purchase a hundred micron diameter glass capillary needle, or make one yourself with a Sutter P 97 pipette Puller, then aspirate sterile saline to fill the dead space in the tubing.

Attach a glass capillary tube to the end of the sterile plastic tubing, eject some saline through the needle to ensure that it is correctly attached without any leakage. If you look under a microscope, see how much pressure is applied and the rate of fluid ejection through the fine tip, we'll use a colored solution to represent the viral vector or stem cells. Place this in a sterile Petri dish, aspirate some air, and then the injection solution.

For our purposes, we use P five mice. In this image, you can see the snout. The location of the eye is covered by an eyelid.

The dotted lines show the lid margin and the lid crease where the incision will be made. Vanis, straight scissors are used to make a one and a half millimeter incision along the closed lid. Fissure pressing forceps are used to pull apart the eyelids and pop to the eye with the lid margin slightly behind the eyeball.

To make the sclerotomy, it's important to be able to identify a few landmarks. The transition from the clear cornea to the sclera is marked by the limbus. Just inside the sclera is the retinal pigment epithelium, then the retina, and then the vitreous Matomy is placed halfway back or at the equator of the globe incision by scratching the surface with a 15 degree microsurgical blade and darkly pigmented animals.

A dark brown pigmented tissue will become visible at the point of incision. If the incision is made too far, you'll actually see fluid come out. This represents the vitreous and it means that you've gone too far.

Passing the RPE retina. This incision could be used for an intravitreal injection. Now, BYO animal.

The incision at this point is complete Additional incision and scratches will release vitreous for a subretinal injection, the goal is to place the micro capillary glass tube into the subretinal space, just between the retina and the RPE. If the incision was too deep, then the needle can be passed past the retina into the vitreous. Insert the needle parallel to the outer wall eye.

To enter a subretinal space. Apply light pressure to the plunger and begin injecting the fluid. If the needle is correctly placed, moderate back pressure will be felt, and a BLE will be created in the subretinal space.

If the needle is in the vitreous, there is significantly less back pressure. To avoid significant reflux, hold a plunger in place for an additional 15 seconds before removing the needle. This will allow the interocular pressure to stabilize.

Gently push the eye back behind the lids into the orbit using curved dressing forceps. Here's another example of a subretinal injection where the dye fills the subretinal space and is clearly visible in an albino animal. The air meniscus allows you to see when you reach the end of your injection.

Fluid injections in a darkly pigmented eye are a little bit more challenging. The careful insertion of the needle will achieve excellent results.