One of the challenges in retinal research is to study the crosstalk between different cells, such as retinal neurons, glial cells, and vascular cells. In particular, isolating and the culturing of retinal neurons is challenging. We believe that cultured retinal explant can overcome this limitation and offer a unique ex vivo system that allow us to study the crosstalk between different retinal cells, under well-controlled biochemical parameters, and independent of vascular system.
Also, a retina explant is a prominent screening tool to study novel pharmacological interventions in various retinovascular and neurodegenerative diseases, such as diabetic retinopathy, which is characterized by vascular and neuronal injury. Our lab has been studying pathophysiological changes, promoting microvascular retinal dysfunction for years. Utilizing a wide variety of models including both in vivo and in vitro models enabled us to optimize a lot of techniques inside our lab.
Here, we describe a detailed protocol for retinal explants derived from a wild-type adult mouse that can be kept in culture for two weeks. Keep the animals housed under constant temperature and light-controlled environment. Inspect every animal for health, adequate food, and water.
Make sure to provide the animals with fresh food and clean water. Change soiled cages on a daily basis or as needed for the health of the animals. Use male black 6 mice of more than 12 weeks of age.
Euthanize the animals in their home cage by carbon dioxide overdose inhalation. After completing the procedure, confirm the animal death by an appropriate method, such as confirming cardiac and respiratory arrest or observing fixed and dilated pupils of the animal. Follow death confirmation by a secondary method of euthanasia such as cervical dislocation.
Apply pressure on the orbit using curved forceps until the globe protrudes. Gently close the forceps at the posterior portion of the eye and elevate in a continuous motion. Immerse the eyeball in a 1.5-milliliter tube containing a one-milliliter ice-cold HBSS.
Transfer the eyeball to a 1.5-or two-milliliter tube containing complete media. Now it is ready for dissection. This diagram illustrates the consecutive steps that will be shown for dissecting the mouse eyeball to create the retinal explant.
Circumferential incision is made along the limbus to open the eyeball. The cornea is removed via dissecting along the limbal incision. The lens and the vitreous are then removed, leaving an empty eye cup.
Via holding the region of the optic nerve head with the fine forceps, peeling off the outer coat of the eye is performed gently. Great attention during the removal of the outer coat in order not to injure the neural retina and have the retina totally intact. Then, the retina will be cut into smaller pieces.
This will be a great advantage, because it will allow you to have an experiment where your control group and experimental groups are derived from the same animal. However, cutting the retina into small pieces will make handling of it to be more challenging. Therefore, we developed a technique for handling these small retinal pieces to ensure proper orientation of the retina culture.
Go with the dissecting scissor blade open just beneath the retinal piece. Slowly elevate the retinal piece with the tip of an open scissor blade and gently transfer it to the culture plate. The explants will be separately transferred onto cell culture inserts in multi-well plates, each of which contain 300 microliters of retinal explant media with the neural retina side facing up.
Retinal explant cultures will be maintained in humidified incubators at 37 degrees Celsius and 5%carbon dioxide. Make sure that the retinal surface is facing upward. If the retinal piece is flipped or folded, try to adjust it gently to the right orientation.
Failure to place the retinal explant in the right orientation with the neural retina facing upward will result in failure of the proper growth of the retinal cells in culture. Here, we can see immunofluorescence staining the retinal explants stained in culture for two weeks. The explants were fixed and stained with NeuN for retinal neurons, GFAP for glial cells, and lectin for blood vessels.
The staining showed well-developed retinal cells and retinal vessels. Here, we can see immunofluorescence staining of retinal explants fixed and stained with GFAP for glial cells and lectin for blood vessels. The images show defective staining and underdevelopment of both retinal cells and retinal vessels.
This is because the retinal explant was folded and was not oriented correctly on the culture plate. Great attention should be taken for the proper orientation of the explant with the retina facing upward. Having the control group being derived from the same single animal as the treated group makes it of great advantage to have more reliable comparison of different experimental conditions.
Retinal implants can provide a decent platform for investigating possible therapeutic targets for a wide variety of retinal diseases, such as diabetic retinopathy and degenerative retinal diseases.
