魚の目を通して糖尿病の勉強: 大人 tg(fli:EGFP) ゼブラフィッシュ網膜血管系の解析、可視化、レーザーマイクロダイ セクション

The overall goal of this dissection protocol for the Adult Zebrafish Retinal Vasculature is to provide a fast read out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes via fluorescence or laser scanning confocal microscopy. This method can help to answer key questions in the field of microvascular complications such as the development of diabetic retinopathy. The main advantage of this technique is that the entire retinovasculature can be presented and analyzed in physiological and disease processes without any application of intervascular fluorescence in a relatively fast and standardized way.

To begin, transfer six milliliters of freshly prepared four percent PFA PBS solution per sample into the wells of six well plates. After euthanizing adult zebrafish according to the text protocol, place them on fresh paper towels and dry them. Use a scalpel and cut the heads behind the operculum.

Then transfer the heads directly into the wells of freshly prepared fixative. Store the plates containing the fish heads at four degrees Celsius for at least 24 hours to assure the fixative penetrates the deeper retinal layers. With heated two percent agarose, fill a Petri dish up to a third and wait until the agar is firm.

Cover the agar plate with one X PBS to create a workspace to dissect the eyes. Transfer the fixed sample into the Petri dish. And by holding the head at the cut surface with one tweezer, insert another, pinned together tweezer below the eyeball at the orbital cavity.

Slowly open the tweezer below the eye and grip the optic nerve. Then carefully tear and detach the eye. To remove any of the four rectus and two oblique extraocular muscles connected to the eye as well as residual extraocular tissue connecting the eye to the orbital cavity, hold back the eye through a half closed tweezer and gripping the structure with the other tweezer use a diametric movement to softly rip it off.

Next, use a 27 gauge disposable needle to puncture the cornea at the outer range. Through this opening use both tweezers to hold the cornea and slightly tear it open. Then carefully work to create a centered tear about the size of the respective pupil.

Apply pressure on the corneal side of the ocular globe at the outer corneal edge above the iris. This will create a small dent and push the lens to the height of the corneal tear. Then run the tweezers under the lens and remove it.

Next, turn the eye upside down with the optic nerve facing upwards. Note that the sclera and cornea are connected and form the fibrous tunic of the eye bulb to protect the cup-shaped retina. This shell, referred to as the corneosclera spares out the area around the optic nerve.

Insert a needle at this discontinuation to create an opening between the sclera and the retina. Then using this access with both tweezers carefully rip the sclera axially into strips to increase the opening around the optic nerve. Be careful to keep the corneosclera intact at the transition to the corneal side circumference.

Before removing the corneosclera from the remaining intraocular tissue try to severe any connections as attachment to other structures will be a critical point. Then hold the sclera with one tweezer and by grabbing the optic nerve with the other and pulling away, fully remove the corneosclera from the eye and discard it. This step will provide a cup-shaped structure consisting of the uvea and retina containing the retinal vasculature.

Next, create a rupture in the choroidal and RPE layer by holding a 27 gauge needle sideways to the remaining cup while scrapping the outer surface with the rim of the needle tip. Use the rupture as an access point to get a grip on the choroidal and RPE layer and use both tweezers to rip it into stripes keeping the connection to the iris intact. Afterwards, run one tweezer under the iris and move it around in a circuit from the outside while creating tension by pulling at the discontinued choroidal and RPE layer and detach the combined structure.

If parts of the iris cannot be removed and stay connected to the cup-shaped retina after removal of the choroidal and RPE layer utilize another natural breaking point that the adult zebrafish eye exhibits inside the photoreceptor layer. In a similar fashion is just demonstrated to remove the iris. Scrape over the remaining cup-shaped retina to induce discontinuations in the photoreceptor layer.

Then use the created access to remove the layer while keeping the possible connection to the iris intact. Afterwards, run the tweezers under the iris and continue in a circuit as demonstrated earlier in this video to detach the combined structure. It's crucial to disconnect the iris from its underlying tissue in a sensible way as it will block fluorescence directly above the inner optic circle during visualization of the vessels.

An overly direct approach can lead to vessel breakage. Then use microdissection spring scissors with a straight two point five millimeter cutting edge to cut the optic nerve as close as possible to the retina. This will allow for better flat mounting of the tissue.

To mount and visualize the retinal vasculature use one X PBS to wash the dissected retina two times for five minutes each. Place a drop of PBS onto a glass slide. Then using a lab spatula with a microspoon end, transfer the retina into the droplet.

With a tweezer keep the tissue in place while using a scalpel to cut the cup-shaped structure to create a flat four petal or five petal shape, depending on the retina size. With a fine piece of paper suck up the left over PBS, taking care not to touch the retina. Then coat the flat mounted retina in mounting medium and cover it with the coverslip.

Be attentive not to create foam as air bubbles can distort visualization of the retinal vessels. Use nail polish to seal the cover. Finally, carry out fluorescence or laser scanning microscopy to visualize the retinal vasculature.

Shown here is a morphological example of the retinal vasculature in adult transgenic fly EGFP zebrafish visualized with a fluorescence microscope and a second example imaged with a confocal laser scanning microscope that reduces background fluorescence. The revealed retinal structure shows a highly organized pattern. The optic artery penetrates the retina at the optic nerve head and in most samples spreads into five to seven main vessels.

The main vessels then branch into a succession of arcades and connect to the inner optic circle or IOC, also called the circumferential vein, encircling the optic disk in the periphery of the flat mounted retina. The adult Zebrafish retina is composed of the following layers from the inside out. The ganglion cell layer, the inner plexiform layer, the inner nuclear layer, the outer plexiform layer, the outer nuclear layer, the photoreceptor layer, and the retinal pigmented epithelium.

The estimation of vasculature parameters from retinal vasculature visualization is shown here. The inner retinal vasculature is situated on the ganglion cell layer while the cordial capillaries would be associated with the retinal pigmented epithelium. Once trained, this technique can be done in under 20 minutes if it is performed properly.

When attempting this procedure, it is important to remember to practice regularly as prolonged absence from preparation reduces the outcome of vessel integrity. Following this procedure, diabetes and neurovascular pathologies can be analyzed in adult zebrafish.