Name: assessment and characterization of hyaloid vessels in mice
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This work was supported by National Institutes of Health (NIH) grants (R01 EY024963 and EY028100) to J.C. Z.W. was supported by a Knights Templar Eye Foundation Career Starter Grant. The hyaloid isolation procedure described in this study was adapted with modification from protocols generously shared by Drs. Richard Lang, Toshihide Kurihara, and Lois Smith, to whom the authors are thankful.

All animals were treated in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research for animal experiments, following the Guidelines of the National Institutes of Health (NIH) regarding the care and use of animals for experimental procedures and the regulations set forth by the Institutional Animal Care and Use Committee (IACUC) at Boston Children&#8217;s Hospital. Lrp5-/- mice (stock no. 005823; Jackson Laborat...

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Techniques to assess and characterize hyaloid vessels are intuitive and necessary procedures to observe the hyaloid vessel regression in animal models, to allow studies on the mechanisms underlying the vascular regression during development. While the in vivo retinal imaging allows the longitudinal observation of hyaloid regression in the same animal, access to a rodent fundus imaging system for OCT and FFA may be a limiting factor. In addition, in vivo imaging in live mice is not feasible before they open their eyes. Th...

The blood supply in the eye is essential to ensure the normal development of the retina and surrounding ocular tissues and to equip a proper visual function. There are three vascular beds in the eye: the retinal vasculature, the choroid, and a transient embryonic circulatory network of hyaloid vessels. The development of the ocular vasculature requires spatial and temporal coordination throughout embryogenesis and tissue maturation. Among the three vascular beds, the hyaloid vasculature is the first functional blood supply system to provide nutrition and oxygen to the newly formed embryonic lens and the developing retina. Hyaloid vessels regress at the same time that the retina vasculatures develop and mature1. The regression of hyaloid vasculature is pivotal to allow a clear visual pathway for the development of visual function; hence, this vascular regression process is as important as the growth of retinal vasculature. Impaired hyaloid regression may lead to eye diseases. Moreover, the regression of hyaloid vessels provides a model system to investigate the cellular and molecular mechanisms involved in the regulation of vascular regression, which may have implications for the angiogenic regulation in other organs as well.
The hyaloid vasculature, derived from the hyaloid artery (HA), is composed of vasa hyaloidea propria (VHP), tunica vasculosa lentis (TVL), and pupillary membrane (PM). It provides nourishment to the developing retina, the primary vitreous, and the lens during embryonic development2. Arising from the HA, VHP branches anteriorly through the vitreous to the lens. The TVL cups the posterior surface of the lens capsule, and anastomoses to the PM, which connects to the anterior ciliary arteries, covering the anterior surface of the lens2,3, resulting in the formation of a network of vessels in the PM3,4,5. Interestingly, there are no veins in the hyaloid vasculature, and the system makes use of choroidal veins to accomplish venous drainage.
In the human embryo, the hyaloid vasculature is nearly complete at approximately the ninth week of gestation and starts to regress when the first retinal vessels appear, during the fourth month of gestation2. Beginning with atrophy of the VHP, regression of the capillary networks of the TVL, the PM, and lastly, the HA occurs subsequently2,3. Meanwhile, the primary vitreous retracts and the secondary vitreous starts to form, composed of the extracellular matrix components, including collagen fibers. By the sixth month of gestation, the primary vitreous is reduced to a small transparent canal extending from the optic nerve disc to the lens, called the Cloquet&#8217;s canal or hyaloid canal, and the secondary vitreous becomes the main component of the posterior segment2,3. The hyaloid circulation vanishes mostly at 35 to 36 weeks of gestation, just before birth3.
Unlike humans, in whom hyaloid vasculature is completely regressed at birth, the mouse hyaloid vascular system starts to regress after birth. As the mouse retina is born avascular and retinal vessels develop postnatally, hyaloid vessels regress concurrently from postnatal day (P) 4 and are mostly completely regressed by P216 (Figure 1). The PM disappears first between P10 and P12, and the VHP disappears between P12 and P16, while a small number of TVL and HA cells remain even at P16, and by P21 the hyaloid vascular system regression is almost complete6. In the meantime, retinal vasculature begins developing after birth. The superficial layer of vascular plexus fully extends to the peripheral retina at P7&#8211;P8, the deep layer (located in the outer plexiform layer) develops from P7&#8211;P12, and finally, the intermediate plexus in the inner plexiform layer develops between P12 and P157. As the retinal vasculature develops, it gradually replaces the function of concomitantly regressing hyaloid vessels, providing nutrition and oxygen to the developing eye. The postnatal occurrence of hyaloid vessel regression in mice provides an easily accessible experimental model to observe and study the hyaloid vasculature, as well as the molecular basis governing vascular regression processes under both physiological and pathological conditions8.
Failure of hyaloid regression can be seen in diseases such as PHPV, which is a rare congenital developmental anomaly of the eye resulting from a failed or incomplete regression of the embryological, primary vitreous and hyaloid vasculature9. The mechanisms regulating the regression process of hyaloid vasculature are complicated and broadly studied. One major molecular pathway essential for the normal regression of hyaloid vessels is the Wnt signaling pathway10, as genetic mutations in this pathway affecting both Wnt ligand and receptors have been linked with PHPV in humans9. Experimental studies identified a Wnt ligand, Wnt7b, which is produced by macrophages around hyaloid vessels in the developing eye to mediate this regression process. Wnt7b activates Wnt signaling by binding with the receptors frizzled4 (FZD4)/LRP5 in adjacent endothelial cells to initiate cell apoptosis, leading to the regression of hyaloid vessels10. As a result, Wnt7b-deficient mice show a persistence of hyaloid vessels10. Similarly, a nonconventional Wnt ligand, Norrin (encoded by the Ndp gene), also binds to FZD4/LRP5 to induce the hyaloid vessel regression during development. Ndpy/-, Lrp5-/-, and Fzd4-/- mice all display postponed hyaloid vessel regression, supporting a critical regulatory role of Wnt signaling11,12,13,14,15,16. Moreover, another Wnt coreceptor LRP6 overlaps with LRP5 in their function on modulating the Wnt signaling pathway in hyaloid vascular endothelial cells17. Other factors that may also contribute to hyaloid regression include the hypoxia-inducible factor18,19, vascular endothelial growth factor20,21, collagen-1822,23, Arf24, angiopoietin-225, and bone morphogenetic protein-426. In this paper, we use Lrp5-/- mice as a model of persistent hyaloid vessels to demonstrate the techniques of assessing and characterizing hyaloid vasculature through both in vivo and ex vivo methods.
The visualization of hyaloid vasculature in vivo and ex vivo is essential for studying the mechanisms of hyaloid vessel regression. Current methods to observe hyaloid vasculature mainly focus on visualizing and analyzing the VHP and HA, through OCT and FFA images, eye cross sections, and the hyaloid flat mount. OCT and FFA are powerful in vivo imaging tools, allowing longitudinal observation in live animals after they have opened their eyes. Moreover, isolated hyaloid flat mount provides visualization of the whole hyaloid vasculature and a means to achieve an accurate quantification of the vessel numbers. Yet the delicate and fragile nature of hyaloid vessels and the resulting technical difficulties of its isolation may have limited its use in eye research somewhat10,17,27. In this paper, we provide a detailed protocol of the visualization of hyaloid vessels, combining both in vivo live retinal imaging and ex vivo isolated hyaloid flat mount to enhance the feasibility of these techniques. This protocol has been adapted with modification and expansion from previous publications on the in vivo method of live fundus and OCT imaging28 and the ex vivo method of isolated hyaloid flat mount11.

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			<td height="21" style="height:21px;width:355px;">AK-Fluor (fluorescein injection, USP)</td>
			<td style="width:191px;">Akorn</td>
			<td style="width:156px;">17478-253-10</td>
			<td style="width:296px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Anti-CD31 antibody</td>
			<td style="width:191px;">Abcam</td>
			<td style="width:156px;">ab28364</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Antifade mounting medium</td>
			<td style="width:191px;">Thermo Fisher</td>
			<td style="width:156px;">S2828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antifade Mounting Medium with DAPI</td>
			<td style="width:191px;">Vector Laboratories</td>
			<td style="width:156px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Artificial tear eyedrop</td>
			<td style="width:191px;">Systane</td>
			<td style="width:156px;">N/A</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:355px;">Bovine serum albumin (BSA)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">A2058</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:355px;">C57BL/6J mice</td>
			<td style="width:191px;">The Jackson Laboratory</td>
			<td style="width:156px;">Stock NO: 000664</td>
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			<td height="21" style="height:21px;width:355px;">Calcium chloride (CaCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">C1016</td>
			<td></td>
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			<td height="21" style="height:21px;width:355px;">Cryostat</td>
			<td style="width:191px;">Leica</td>
			<td style="width:156px;">CM3050S</td>
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			<td height="21" style="height:21px;width:355px;">Cryostat</td>
			<td style="width:191px;">Leica</td>
			<td style="width:156px;">CM3050 S</td>
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			<td height="42" style="height:42px;width:355px;">Cyclopentolate hydrochloride and phenylephrine hydrochloride eyedrop</td>
			<td>Cyclomydril</td>
			<td style="width:156px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">G9382</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;width:355px;">Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:156px;">A-11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating board</td>
			<td style="width:191px;">Lab-Line Instruments Inc.</td>
			<td style="width:156px;">N/A</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:355px;">Isolectin GS-IB4, 594 conjugate</td>
			<td style="width:191px;">ThermoFisher Scientific</td>
			<td style="width:156px;">I21413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Ketamine hydrochloride injection</td>
			<td style="width:191px;">KetaVed</td>
			<td style="width:156px;">NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Lrp5-/- mice</td>
			<td style="width:191px;">The Jackson Laboratory</td>
			<td style="width:156px;">Stock NO. 005823</td>
			<td>Developed by Deltagen Inc., San Mateo, CA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Micron IV and OCT</td>
			<td style="width:191px;">Phoenix Research Labs</td>
			<td style="width:156px;">N/A</td>
			<td>Imaging software: InSight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Microscope</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:156px;">discovery v8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Microsurgery forceps</td>
			<td style="width:191px;">Scanlan International</td>
			<td style="width:156px;">4004-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Microsurgery scissors</td>
			<td style="width:191px;">Scanlan International</td>
			<td style="width:156px;">6006-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Optimal cutting temperature compound</td>
			<td style="width:191px;">Tissue-Tek</td>
			<td style="width:156px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Optimal cutting temperature compound</td>
			<td style="width:191px;">Agar Scientific</td>
			<td style="width:156px;">AGR1180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde (16%)</td>
			<td>Electron&#160;Microscopy&#160;Sciences</td>
			<td style="width:156px;">15710</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;width:355px;">Peel-A-Way disposable embedding molds (tissue molds)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:156px;">12-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-buffered saline (PBS) buffer (10X)</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:156px;">P0496</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Slide cover glass</td>
			<td style="width:191px;">Premiere</td>
			<td style="width:156px;">94-2222-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost microscope slides&#160;</td>
			<td style="width:191px;">Fisherbrand</td>
			<td style="width:156px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Triton X-100</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:156px;">X100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:355px;">Xylazine sterile solution</td>
			<td style="width:191px;">Akorn: AnaSed</td>
			<td style="width:156px;">NDC: 59399-110-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment and characterization of hyaloid vessels in mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

In vivo imaging of hyaloid vessels in live mice 
Figure 3A reveals cross-sectional views of OCT images for the retina and hyaloid tissues in 3-month-old WT and Lrp5-/- mice, an animal model with persistent hyaloid. The WT eye shows the absence of hyaloid tissue, whereas the Lrp5-/- eye shows two persistent hyaloid vessels derived from the optic nerve head. Figure 3B d...

Watch this Scientific Journal Video about Assessment and Characterization of Hyaloid Vessels in Mice at JoVE.com

Assessment and Characterization of Hyaloid Vessels in Mice

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...

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.

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