This procedure starts off with a well perfused monkey head. With the brain removed, the eyeballs are then extracted and connected. Tissue cleared away a next, the cornea lens, sclera and vitreous humor are carefully removed, freeing the retina.
The retina is placed flat on a glass slide, and the remaining vitreous humor is eliminated. Iso Dentrix samples are then taken and cryo protected. Hi, I am Dr.Mark Burke from the Visual Neurosciences Laboratory at the School of Optometry at the University of Montreal.
Hi, I'm Dr.Shaheen Zur, also from the Visual Neurosciences Laboratory in the School of Optometry at the University of Montreal. Hello, I'm Dr.Tito, director of the Visual Neuroscience Laboratory at the School of Optometry, university Of Montreal. Today we will show you a procedure for dissecting a non-human primate retina.
We use this procedure in our laboratory to study various aspects of retinal organization in normal development and in response to developmental intrusions. So let's get started. Start this protocol with the primate head, which you have previously perfused.
Well, with a fixative such as Paraform aldehyde, the retina to be dissected here is harvested from a vervet monkey. To ensure preservation of the eye, a fixative such as paraform aldehyde can be injected just under the lens. The head should be stored in fixative or PBS before moving on to retrieving the eyeballs to gain easier access to the eyeball.
First, remove the brain. To view this procedure, consult our recent JoVE article dissecting the non-human primate brain in stereotaxic space. With the brain removed the thin walled orbit bone is readily apparent.
Use bone urs to slowly chip away the wall of the orbit. Use a scalpel to cut away the ocular muscles and remove the connective tissue from the eyeball. Finally, carefully cut the optic nerve releasing the eyeball from the orbital cavity.
Now that the eyeball is released, the retina can be removed To remove the retina, first, place the eye in a Petri dish filled PBS to keep the retina from drying. Start by removing the cornea. Use a pair of spring scissors and cut the sclera close to the perimeter of the cornea at the level of the ora serrata.
Using forceps, remove the lens. Finally, to remove the retina from the sclera. Use a paintbrush, forceps, and spring scissors.
Separate the retina from the sclera and then cut the sclera away with the scissors. One must carry out this process in small increments to avoid damaging or tearing the retinal tissue. The sclera is not readily separated from the remnant optic nerve.
So carefully cut the sclera around the optic nerve. The retina maintains its curved shape. Remove the jelly-like vitreous humor in a lump.
The retina is now ready for mounting and sampling. In order to flatten the retina onto a slide, make several radial cuts with a scalpel blade. Use ordinary filter paper and a paintbrush to remove the residual vitreous humor.
It is imperative to be gentle while performing this step as it exposes the retinal ganglion cell layer. Check if the optic nerve is still attached at the optic disc. Use a scalpel blade and a pair of spring scissors to remove the optic nerve without ripping the retina.
Now flat mount the retina on a two by three inch glass microscope slide with a retinal ganglion layer away from the slide. The fovea is now apparent as a dark patch in the temporal ventral direction from the optic disc. To examine the retinal ganglion cell layer, the pigmented epithelium may need to be removed or bleached.
Use a paintbrush to lightly remove some of the pigmented epithelium. Note that this method tends to damage the photoreceptor layer. Examining the cell distribution and morphology of each layer throughout the retina requires isometric sampling of the retina.
Start by flat mounting the retina on a slide and add PBS to keep the retina moist. Then cut small pieces of tissue equidistant from the optic disc in the nasal, temporal, upper and lower directions. These cut pieces can now be sectioned in the coronal plane with a cryostat vibrato or ultra microtome.
Depending on the research question. For cryostat sectioning, the pieces should be cryo protected in 30%sucrose in phosphate buffer overnight at four degrees Celsius. Following the overnight incubation, freeze the retinal pieces in a mounting medium on dry ice.
Use the cryostat to yield sections as thin as four microns. In our laboratory, we routinely perform immunohistochemistry on cryo sectioned retina. In this case, we are interested in the iso density of cannabinoid receptors in the primate retina.
We also examine the effects of prenatal ethanol exposure on the primate visual system. To this end, we are interested in cell density and layer thickness in the fovea and in the peripheral retina. To accomplish this, we embed pieces of retina in epon slice at 700 nanometers on an ultra microtome and stain with 1%toine.Blue.
We've just shown you how to dissect a primate retina When doing this procedure. It's important to remember not to damage the retinal ganglia, cell layer or the photoreceptor layers. So that's it.
Thanks for watching and good luck with your experiments.
