Retinal Cup Extraction from Mouse: A Surgical Procedure to Obtain Intact Retinal Cup from Mouse Eye

Overview

This video describes a protocol to isolate the retinal cup without the outer covering layers from the eyeball of a mouse. The extracted retinal tissue can help study various metabolic pathways occurring in the retinal cells.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Retinal cup dissection

1. Euthanize a mouse by CO₂ asphyxiation following institutional guidelines on euthanasia.
   NOTE: Do not leave the animal in a CO₂ chamber longer than the time needed for euthanasia.
2. Enucleate eyes and place into ice-old 1x PBS buffer in a petri-dish and then place it under a dissection microscope.
3. Carefully remove, by cutting with microscissors, the extra rectus muscles attached outside the eyeball and cut off the optic nerve.
4. Use a 30 G needle to punch a hole at the edge of the cornea (limbus); this serves as the insertion site for the microscissors. Then, use a fine dissection microscissors to make a circular cut along the edge of the cornea, separating it from the posterior eye cup.
5. Use sharp dissection forceps to remove the cornea, lens, and the vitreous humor away from the eye cup.
6. Use fine dissection microscissors to make several small cuts on the scleral layer at the rim of eye cup. Avoid cutting the retina layer. Use two sharp dissection forceps to hold on to the scleral tissue at each side of the cut and very carefully pull on the scleral layer to remove it from the neural retina. Repeat this around the eye cup until all sclera is removed and an intact retinal cup is obtained.

Materials

Name	Company	Catalog Number	Comments
1X PBS	Thermo Fisher	14190-144
30-gauge needle	BD Precision Glide	305106
Biopsy puncher, 1 mm	Integra Miltex	33-31AA
CO2 asphyxiation chamber
Dissection forceps-Dumont #5	Fine Science Tools	11251-10	Stright tip
Dissection forceps-Dumont #7	Fine Science Tools	11274-20	Curved tip
Dissection microscope
Graefe forceps	Fine Science Tools	11051-10	Curved, Serrated tip
Microscissors	Fine Science Tools	15004-08	Curved tip