Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The overall goal of this procedure is to inject retinal pigment, epithelium or RPE cells into the subretinal space of rodents while minimizing trauma. This is accomplished by first using a sharp needle to poke a hole in the eye of a sedate animal. Next, the cells are injected into the subretinal space using a blunt needle.

In the final step, the eye is coated with moisturizer to keep it hydrated. Ultimately, histology and in vivo imaging modalities can be used to monitor the effectiveness of the injection. While the method we've shown you today was specifically tailored for the delivery of RPE cells into the subretinal space, the technique itself is broadly applicable to delivery of a variety other cell types and chemicals into the subretinal space to be used in a variety of preclinical studies.

Generally, individuals that are new to this method will struggle because it's hard to see how well the injection was performed. Visual demonstrations of this method are critical as the injection steps are difficult to learn without seeing them performed. In person.

About an hour before the injection prewarm a non trypsin cell dissociation solution, sterile PBS and culture media to 37 degrees Celsius disassemble and place the components of a syringe with a 33 gauge blunt needle in boiling water. Next, detach the RPE cells in the cell dissociation solution for five to eight minutes. At 37 degrees Celsius, gently scraping to release any cells that are still attached and then dilute the cells in 15 milliliters of culture media to inactivate the enzyme, transfer the dissociated cells into a 15 milliliter tube, and then after counting, spin down the cells and resuspend the pellet at two times 10 to the fifth cells per microliter in sterile prewarm.

PBS then transfer the cells into a 1.5 milliliter micro centrifuge tube, and load the sterilized syringe with 0.5 microliters of cells for immediate injection to inject the cells After confirmation of sedation by toe, pinch, place the anesthetized rodent on its side so that the eye that will be injected is facing up. Then under a dissecting microscope, hold the animal's head with two fingers just above the ear and by its jaw and gently stretch the skin parallel to the eyelids so that the eye pops slightly up out of the socket. Next, holding it at an angle.

To avoid touching the lens and taking care to avoid any vessels, use the 30 gauge sharp needle to make a hole immediately below the limbus Injections. Work better with two people so that one person can pass the injector, the blunt needle, and the syringe, allowing the injector to maintain focus on the mouse's eye. Now retract the disposable sharp needle from the eye while maintaining the grip on the head.

Taking care to remember exactly where the hole is and mount the preloaded syringe. Equipped with the blunt needle on a micro manipulator, insert the tip of the blunt needle through the hole. Taking care, again, not to touch the lens and gently push the needle through the eye until resistance is felt.

Keeping all movements to a minimum. Carefully inject the RPE cells slowly into the subretinal space. RPE Retina detachment will occur.

However, a cleaner injection minimizes the detachment and greatly improves the chances of reattachment, then retract the syringe slowly and apply eye moisturizing drops to keep the eye hydrated, monitoring the animal until it is fully recovered. RPE cells can be delivered into the subretinal space of rodents quickly and consistently. Using this technique.

While not required, traumas can be greatly minimized using a micro manipulator. With this setup when performed cleanly fluorescence from labeled RPE cells can be detected using a confocal scanning ophthalmoscope and the induced retinal detachment can be visualized using an optical coherence tomography system. Confocal scanning Ophthalmoscopy was used to capture these images around the entire injection site immediately following the subretinal injection.

Note, the detachment that is the most profound but not severe in the center images Once mastered, the actual injection can be performed in five minutes if it's done properly While attending this procedure, it's important to work carefully and keep unnecessary movements to a minimum. After watching this video, our hope is that you'll have a good understanding of and appreciation for the technique of subretinal injection into rodents. We hope that this will be effective way to deliver cells, the same time minimizing discomfort and trauma to the eye of the rodents.