Ocular research in the earlier times mostly relied on the ocular tissue from deceased individuals. However, over the years, many ophthalmic rodent models have been developed and the need for human ocular tissue has diminished. Due to the small size and the limited area of operations in the rodent's eye, the effective access to the ocular subparts is severely constrained.
Typically, to operate on a rodent's eye, the eye is immobilized via the optic nerve and the dissection is performed. This practice is arduous and can damage the fragile tissue as the spherical eye continues to move during the dissection. Despite being beneficial in isolating the various sections of retinal layers, this technique cannot demarcate the spatial orientation of the tissue upon dissection of the tissue.
Further, a targeted intervention is difficult to locate post dissection due to the homogenous cellular assembly of the posterior and anterior eye cup. In this video, a modified surgical method for microdissection is demonstrated. The presence of the attached nictitating membrane, or the third eyelid, presents unique and significant advantages.
In this method, first the eyeball is enucleated with the third eyelid, then the third eyelid is used to immobilize the eye. It is followed by piercing the eyeball through the corneal limbus, and the incision is used as the point of entry. Then, by cutting along the circumference, anterior and posterior eye cups are separated.
By dissecting the posterior eye cup further, the translucent layer of the neural retina is identified and gently peeled off. The neural and RPE layers are thus obtained for further processing. The third eyelid will enable us to demarcate the spatial orientation of the eye post enucleation.
Let us now follow detailed step-by-step procedure. Dilate the rodent eyes using a drop of 0.8%tropicamide and 5%phenylephrine ophthalmic solutions and place the animal in a dark area for 30 minutes before the procedure. Euthanize the animal by intraperitoneally administering five times doses of anesthesia.
A typical dose is 200 microliter PBS containing 10 mg ketamine and one mg xylazine. Confirm complete lack of response to stimulus by examining the pedal reflex. Place the mouse in lateral recumbency to allow complete access to the eye.
Keep a Colibri suturing forceps, a curved forceps, a micro-blade 15 degrees three millimeter size, spring micro-scissors, and an angled spring micro-scissors ready for the procedure. Place a curved forceps around the eyeball and pinch to free the eye from extraneous attachments. The third eyelid, also known as the nictitating membrane, is located towards the upper corner of the nasal tangent and can be identified as a minor translucent protuberance with pigmented boundaries.
Next, use a spring micro-scissor and cut around the eyeball. Small, continuous cuts release the eyeball from scleral attachments. Place a curved spring micro-scissor under the eyeball to free the eye by cutting the optic nerve.
Place the enucleated eye in a Petri dish containing PBS buffer such that the eyeball is submerged in PBS. Some extraocular muscles or tissues will begin to float away from the eyeball. Remove them by cutting them off the eyeball using spring micro-scissors.
Then, remove the optic nerve from the base of the eyeball to allow better separation of retina for wholemount preparations. It is not necessary to remove the optic nerve for tissue sectioning. Transfer the eyeball into a 1.5-ml tube containing one ml of 4%paraformaldehyde or PFA.
Keep it for two hours at four degrees Celsius for tissue fixation. It is possible to pause at this step by leaving the tissue in PFA for up to 12 hours at four degrees Celsius. However, for the isolation of live cells, the fixation steps should not be performed and the entire procedure needs to be performed in continuum.
After PFA fixation, transfer the eyeball into a Petri dish containing PBS and place it under a dissecting microscope to perform the subsequent procedure. Immobilize the eyeball by holding down the third eyelid firmly with Colibri suturing forceps. The limbus is the corneal scleral junction.
Make a small incision at the limbus by piercing it using a microblade. The incision is made preferably opposite the third eyelid. Next, using this incision as a starting point, with the micro-scissor make small and continuous cuts along this corneal scleral limbus circumference.
First, complete a semicircle of cuts starting from the incision point till the third eyelid. Then complete the remaining semicircle of cuts on the other side. Finally, make a cut around the third eyelid to separate the posterior and the anterior cup.
Remove the lens from the posterior cup using forceps to obtain the posterior cup comprising of neural retina and RPE layer and the corneal anterior cup. If working with unfixed tissues, the anterior and posterior cups can now be enzymatically digested to obtain corneal or retinal single-cell suspension. For fixing the tissues, transfer these cups into a 1.5-ml tube containing 1 ml of 4%PFA for 12 hours at four degrees Celsius.
Post fixation, the tissue can be embedded in appropriate medium and cryosectioning or paraffin sectioning can be done to obtain corneal or retinal sections. Reintroduce the posterior cup into the Petri dish containing PBS buffer. An opaque layer on the pigmented RPE layer is the neural retina.
Peel off the neural retina using a second forceps. A very gentle and slight movement of hand is highly recommended to not damage the tissue. At the optic nerve, exit, the curved spring scissors can be inserted under the neural retina, and a cut can be made to completely free the layers if the neural retina remains attached.
The retina can also be freed or detached by gently stroking it with a soft brush. Reintroduce the anterior and posterior cups into the Petri dish containing PBS buffer. Use an angled spring micro-scissor to make a cut next to the third eyelid from the periphery perpendicularly towards the optic center.
Maintain a grip of the third eyelid and make a cut next to the first cut. Make a similar cut between the second cut and the third eyelid. Three or four such equidistant cuts open the hemispherical cups into a flower shape, which falls flat and can be easily mounted.
Do not make very deep cuts until the center of the cup, as this may make the leaf sections to fall apart or separate easily. The liquid medium allows the anterior eye cup to attain a curved structure. Follow the same steps as given for making cuts on the posterior cup to make three or four equidistant cuts on the anterior cup.
The tissues are now ready for further staining. In this figure, the wholemount of anterior cup is shown. The corneal tissue was microdissected as described to reveal the lymphatic vessels in the attached conjunctival tissue of the corneal leaflet.
In this figure, the wholemount of neural retina is shown. The retinal vasculature of the neural retina was stained with Isolectin and is visible as green vessels. In this method, the enucleated eyeball of the mice is obtained with the attached third eyelid for effective and easy handling.
The third eyelid immobilizes the eyeball completely and allows the dissection to proceed with ease and minimum errors. This method is advantageous for ocular tissue-specific sectioning, single-cell analysis, and wholemounts. However, the need for repetitive tissue fixation renders the tissue unviable for cell culture or such live cell necessities.
Hence, the procedure needs to be performed in continuum for live cell isolation.
