網膜色素上皮の全マウントするためのマウスの眼の解剖

My name is Allison Clavon. I'm a research assistant for Dr.Alex Bishop at the Gree Children's Cancer Research Institute at UT Health Science Center in San Antonio, Texas. Today I'm going to demonstrate how to dissect a rodent eye for a whole amount of the retinal pigment epithelium.

This tissue is of interest for studying retinal development and diseases and is also the site of an in vivo homology directed DNA repair assay. So let's get started. Here is an eye ready for dissection.

First, let's take a look at the anatomical structures you'll use as landmarks throughout the procedure. This eyes from a dilute animal. Here is the optic nerve at the back of the eye opposite.

That is the cornea. The iris shows through the cornea. The whitish area at the back of the eye is the sclera.

Therefore, this region is called the cornea scleral divide. The first step of dissection is to remove extraneous tissues such as muscle connective tissue and the optic nerve. Next, the cornea must be removed, making a circular cut.

At this point, the lens can be popped out resulting in a hollow eye cup. Next four cuts are made from the corneal scleral divide toward the optic nerve head. The resulting structure resembles a four pedaled flower.

Each of these four petals is then cut down the middle, resulting in a structure that resembles an eight pedaled flower. At this stage, the I may buckle in areas due to its spherical nature. To combat this and achieve a flatter hole mount, you can make eight shorter cuts just passing the buckle.

You'll need several materials handy at your workstation, including a 100 or 200 p pipette and corresponding tips, one X-P-B-S-A 35 millimeter dish slides and cover glass, 90%glycerol and clear nail polish for slide preparation, a rack for drying slides and a box of Kim wipes and a waist disposal container. In addition to these common materials, you'll also need a set of specialized microdissection tools, including straight forceps, 45 degree angled forceps are used to hold, turn, and generally manipulate the eye throughout dissection. These 15 degree forceps have an angle up and are also used for handling and manipulation of the eye.

Finally, you'll need a pair of spring scissors. It's very important that you use spring scissors as opposed to traditional style scissors. For this dissection, before you begin dissection, make sure your chair height is adjusted so that when you're sitting up straight, your eyes are level with the oculars.

Slouching at the scope causes shoulder and back pain and inhibits the quality of your dissections. To begin pour one XPBS into the lid of the 35 millimeter dish. Then transfer the eyes you plan to dissect into the dish.

This is an eye from a wild type C 57 black mouse. You can see it as very small about three and a half millimeters in diameter. The opacity of the sclera gives it a bluish hue.

The first step in dissection is to remove all extraneous tissues from around the eye, such as muscle connective tissue and the optic nerve. Here I have the 45 degree forceps in my left hand and the 15 degree forceps in my right hand. You can use one pair of forceps to steady the eye while pulling tissue away with the other.

When a large piece of tissue comes free, carefully wipe your Forceps on a Kim wipe. Just be careful not to crush or puncture The eye. One of the benefits of performing this dissection in suspension is that you can easily see the connective tissue floating away from the eye.

Eventually, you'll reach a point where you can no longer remove any more tissue using forceps. Trade out your 15 degree angled forceps for your spring scissors. Use the spring scissors to clip off the optic nerve.

Use the outside edge of the blade to Push the tissue forward toward the cornea scleral divide. Then trim and gently wipe your scissors on a kim wipe. Let's look at that again.

Push the tissue forward toward the cornea scleral divide with the outside edge of your scissors when the tissue Is sticking up. Trim, continue pushing Bits of tissue forward with the blunt edge of your scissors and then trimming them away until all extraneous tissue is removed. There are usually two bands of tough tissue on the eye.

The first is attached at the corneal scleral divide, and the second shown being trimmed. Here is about halfway between the corneal scleral divide and the optic nerve head. The next step in dissection is to remove the cornea and lens.

Use your 45 degree forceps to pinch up a flap at approximately the middle of the cornea. Then use your spring scissors to make a small incision at the base of the flap. Insert the lower blade of your scissors into the incision under the iris.

Make a cut toward, but not into the Corneal scleral divide.Now. Slowly rotate the eye 360 degrees, making small cuts all the way around. Try to keep the scissors under the iris, cutting both the cornea and the iris at the same time.

It takes a lot of practice to do it as easily As I show it here. Once the cornea is Removed, turn the eye on its side and gently press on the outside of the eye pushing the lens out. The remaining structure will be cup like next.

The eye must be quartered. To do this, turn the eye on its side and insert the lower blade of your spring scissors. Make a longitudinal cut toward, but not all the way to the optic nerve head.

To do this, keep your arms at your side and your scissors in front of you approximately perpendicular to your body and the eye cut. Make your second cut directly opposite the first cut. Take your time lining up so that you can make a smooth cut.

Making multiple cuts can cause jaggedness, but if Lined up carefully are not noticeable, notice how I'm able to Work with this spherical shape of the eye rather than against it because the eye retains its normal shape. When Suspended in PBS, the rest of the dissection will take place on A slide. Cradle the eye with your 45 degree angled forceps and lift it out of the PBS.

Place the eye on a fresh slide. Using both pairs of angled forceps, carefully unfold the eye. Notice there is a small margin of cornea and iris around the perimeter of the eye, which is useful for handling.

Next, you need to cut each of the four pedals in half. Rotate the eye so that one of the pedals is perpendicular to your body. Grasp the corner of the pedal with your 15 degree angled forceps.

Lift up slightly and then make a cut from the cornea scleral divide toward the optic nerve head. These cuts should be perpendicular to a tangent. A tangent is a line that is perpendicular to the diameter.

Therefore, your cuts should be in line with the diameter of the eye. Now rotate the slide so the next pedal is perpendicular to your body and repeat. Grasp the corner of the pedal.

Lift up slightly and cut. Repeat until each of the four large pedals has been cut in half. The whitish substance is the neural retina, which directly overlays the RPE.

The neural retina can protect the RPE, so leave it intact until all of your cuts have been made. Notice how the pedals are slightly curved. Do not fight against this.

Take your time lining up so you can make a good single cut. Because the eye is round, it has a tendency to buckle to prevent this buckling. You can make eight shortcuts.

One on each pedal. Use the same basic strategy as the previous round of four cuts. Lift the corner of the pedal and cut, but in this case, only cut About halfway down the length of the pedal.

Once all your cuts are made, you Can remove the neural retina. Here I'm adding a few drops of one XPBS to the specimen. You don't wanna let your RPE dry out.

The neural retina should only be loosely associated with the underlying RPE. You can see it lifts off quite easily. However, it will usually remain attached at the corneal scleral divide, which on the inside of the eye is a bumpy ridge called the ciliary body.

You can insert the tip of your 15 degree forceps into the optic nerve head to anchor the specimen while pulling off the neural retina. The ciliary body marks the edge of the RPE, the darkly pigmented tissue that came off. Here is iris, not RPE.

I recommend removing as much of the iris as possible because it is very sticky and can fold over and stick to the RPE if the neural retina is flaky or if remnants of it remains stuck to the RPE. One of two things probably went wrong. Either the eyes were not fixed properly, such as for too long or too short a period of time, or they were in storage for too long before dissection.

The pedal I'm uncovering here has not yet had its shortcut. You can see how the pedal about a third of the way from the edge of the RPE. This is why The eight shortcuts are recommended.

Once all of the neural Retina has been discarded, use a pipette to rinse the RPE several times with 100 to 200 microliters of PBS until all dust and debris has been removed. Pipette 80 to 90 microliters of 90%glycerol onto a fresh slide in a rectangular shape. Note, this is the optimal volume of glycerol for a 22 by 50 millimeter cover glass and should be adjusted accordingly.

For other sizes of cover glass, use your pipette tip to spread the glycerol. Be careful not to add air bubbles to the glycerol. If you do get a bubble, drag it to the edge where it will pop.

Slide the lower arm of your 15 degree forceps under the RPE and lift up carefully, slowly lower the RPE into the glycerol. The RPE will float on top of the glycerol and may curl or twist a bit. Use your forceps to straighten the RPE and to drag the glycerol over the surface of the RPE.

If any air bubbles form. As you do this, drag them to The edge where they will pop holding the cover glass at about 45 Degrees. Touch it to the edge of the glycerol.

Then move it toward the edge of the slide. Hold the bottom of the cover glass with your thumb and forefinger and cradle the opposite edge with your 15 degree forceps. Very slowly lower the cover glass toward the slide as soon as contact is made.

With the RPE, stop lowering, allowing the glycerol to migrate continue to lower your cover glass slowly. If you lower the cover glass too fast, air bubbles will become incorporated into your slide here. I will lower the cover glass slowly as it touches the RPE and allow the glycerol to migrate.

You can see when I let the cover glass go quickly, air bubbles become incorporated. Use a generous amount of clear nail polish to attach the edge of the cover. Glass to the slide go all the way around the perimeter.

This is a typical RPE from a C 57 black mouse. It is normal to see underlying vasculature as indicated here. It is also normal to see some variation in pigmentation such as patches that appear to be less pigmented.

However, clear spots such as this are likely to be due to physical damage to the RPE and the underlying choroid. Because the choroid of the eye is also pigmented. It is possible for damage to a black RPE to be unnoticeable if the choroid is left intact.

This is a typical RPE from a dilute mouse. As indicated, you will be able to see vasculature and there will be some variation in pigmentation such as darker patches and lighter patches. However, it is nearly impossible to see damage on a dilute RPE.

Are these small patches damage or are they normal variation in pigmentation? I suggest foid and staining to address this question. It will give you an idea if you're handling the I two roughly.

The stain clearly outlines the cell membranes, making damaged spots obvious, but in all reality, the RPE is fairly resilient, and if you handle the eye by the cornea, you are unlikely to cause damage. Here is a whole mount with a host of problems, the least of which indicated here is too wide a margin of cornea, which can lead to buckling and folding the squiggly material. You can see here is a massive extraneous tissue remaining on the back of the eye.

This pedal is partially folded over, and here you can see the RPE has been partially torn. Another problem is that this eye has the appearance of a pinwheel. Here I have drawn in the angles of the cuts.

However, this is how they should have been. Remember, your cuts should be perpendicular to the tangent and in line with the diameter. I've just demonstrated how to dissect a mouse eye for a whole mount of the retinal pigment.

Epithelium, the most important thing is to maintain good posture, but try to stay relaxed. I hope you found this video useful and wish you all the best with your future experiments.