Our protocol provides both in vivo and ex vivo measures for visualizing and characterizing the hyaloid vasculature within the eye as a useful experimental model for studying vascular regression. This technique combines both in vivo longitudinal observation of hyaloid vessels in live mice and ex vivo visualization and accurate quantification of dissected whole hyaloid vessels. Our technique provides a practical and feasible method for studying the pathogenesis and basic mechanisms of persistent fetal vasculature.
This method also provides insight into cellular and molecular mechanisms involved in the regulation of ocular vascular regression which may be relevant for angiogenesis research in other organs. The hyaloid isolation steps may be technically challenging for beginners. Plenty of practice and patience will help to ensure a successful dissection.
Visualizing this method will provide detailed hands-on information and tips about the technique that may assist viewers in mastering the skills more efficiently. For optical coherence tomography or OCT imaging, adjust the mouse and probe so that the optical nerve head is in center of the visual field in the fundus image and adjust the focus and the angle of the indicated line in the OCT imaging software to reveal the persistent hyaloid vessels before obtaining images. For fundus fluorescein angiography imaging of the hyaloid vessels, adjust the alignment slightly to position the optic nerve head in the center of the view field and change the microscope filter to a green fluorescent channel.
Then focus on the persistent hyaloid vessels and obtain multiple images at one, three, five, and 10 minutes postinjection to determine the best time point for the signal-to-background ratio for observing the vessels. For ex vivo hyaloid vessel dissection and visualization use microsurgery forceps to open the eyelids of a postnatal day-eight mouse and place curved microsurgery forceps under the globe in the orbit of one eye to grasp the optic nerve without squeezing the eyeball. Gently pull and remove the eyeball with forceps and fix the eye in 4%paraformaldehyde at room temperature.
After 30 minutes transfer the fixed eyeballs to ice-cold PBS under a dissection microscope and inject 50 microliters of gelatin solution into the vitreous body of the eyeball at four different evenly spaced sites around the limbus. Then incubate the eyeball on ice for 30 minutes to solidify the injected gelatin within the vitreous space. When the gelatin has cured transfer the eyeball to a Petri dish of PBS under the dissecting microscope and use microsurgery scissors to make an incision at limbus to remove the cornea.
Snip and remove the optic nerve under the dissecting microscope. Using two pairs of forceps peel off and discard the sclera, choroid and retinal pigment epithelium layers, followed by removal of the iris. With the optic nerve side of the retina facing up and the lenses facing down, inject 50 microliters of PBS just beneath the retina cup to allow accumulation of the PBS between the gelatinized vitreous body and the retina.
Use microsurgery forceps to gently peel the retina cup and ciliary body from the vitreous body. Using a transfer pipet transfer the gelatin cup containing the hyaloid tissue immersed in PBS to a microscope slide. And turn the remaining tissues over so that the lens is facing up.
Then lift the lens and gently loosen the connection between the lens tunica vasculosa lentis and the vasa hyaloidea propria before cutting the hyaloid artery with the microsurgery scissors to remove the lens tunica vasculosa lentis. Keep the vasa hyaloidea propria region of the hyaloid cup for the flat mount. For flat mounting of the hyaloid vessel samples gently rinse the hyaloid cup with fresh PBS to remove any dissection debris.
With the tissue floating in PBS use microsurgery forceps to gently arrange and adjust the position of the gelatinized hyaloid cup so that the rim of the cup is facing down. Place the slide with the hyaloid cup immersed in PBS onto a slide warmer at 37 degrees Celsius. After the gelatin melts and the hyaloid flattens remove the slide when it is barely dry and add a drop of anti-fade mounting medium with DAPI to stain the nuclei in the hyaloid vessels.
Then gently place a coverslip on the hyaloid to complete the flat mount. To image the DAPI staining of the hyaloid vessels transfer the mounted sample onto the stage of a fluorescent microscope and confirm that the central hyaloid artery is visible. Then identify the vessel branches directly derived from the hyaloid artery and manually count the number of vessel branches for quantification.
Here, cross-sectional views of OCT images for the retina and hyaloid tissues in three-month-old wild type and low-density lipoprotein receptor-related protein five or LRP5 knockout mice, an animal model with persistent hyaloid vessels, are shown. In these fundus fluorescein angiography images of persistent hyaloid vessels eight branches of hyaloid vessels are observed in the vitreous body of six-week-old knockout mice while no remnant of the hyaloid vessels is observed in wild type animals of the same age. In these hyaloid vessel flat mounts the vascular cells and associated macrophages can be identified by their nuclear DAPI staining.
And each line of cells branching from the central hyaloid artery represents one vessel of vasa hyaloidea propria. Wild type mice have an average of 12 branches of hyaloid vessels at postnatal day eight, while age-matched LRP5 knockout pups exhibit around 25 branches of hyaloid vessels demonstrating a significantly impaired regression of hyaloid vasculature. In addition, delayed and incomplete retina vascular development is also observed in LRP5 knockout pups.
Indeed, hyaloid vessels can still be identified in cross-sections of LRP5 knockout eye tissue sections at postnatal day eight. Whereas the wild type eyes no longer display these vessels. The most important thing to remember is to be gentle and patient when isolating the hyaloid system from the retinal and other ocular tissues.
After isolating the hyaloid vessels immunostaining with different cell markers can be performed to uncover the cellular interactions regulating hyaloid regression. This technique explores the underlying cellular and molecular mechanisms of persistent fetal vasculature in ocular angiogenesis research. Paraformaldehyde, ketamine, and xylazine are hazardous.
Please prepare these reagents in a chemical fume hood using the proper personal protective equipment.
