Quantitation of Hemodynamic Function during Developmental Vascular Regression in the Mouse Eye

Abstract

purpose. Ultrasound biomicroscopy (UBM) utilizes frequencies higher than conventional diagnostic ultrasound and can noninvasively provide anatomic and functional information about mouse ocular structures in vivo at high resolution. Vascular development can also be assessed with high-frequency Doppler imaging, which permits detection and characterization of ocular blood flow not detectable at lower, conventional Doppler frequencies.

methods. The eyes of CD-1 mice were examined daily from the day of birth to postnatal day (P)16. Hyaloid vascular system anatomy was imaged with UBM and microcomputed tomography (microCT). Blood flow velocity was also measured with Doppler UBM imaging in the hyaloid artery, vasa hyaloidea propria, tunica vasculosa lentis, and retina.

results. In the mouse, the hyaloid vasculature degenerated from a well-defined structure at birth by progressive loss of branches. Hyaloid regression coincided with a progressive decrease in blood velocity detected in the hyaloid vascular structures, which is thought to be one of the major triggering factors of the regression in these vessels. At P13, no further blood flow was detected in the CD-1 mouse hyaloid vasculature. An inverse relationship was also shown between peak blood velocity in the lens and retina.

conclusions. UBM imaging provides a valuable means of rapidly and noninvasively characterizing ocular development in vivo. MicroCT scans have also provided intralumenal images of hyaloid vascular structure. This is the first study of vascular structure and function during the dynamic process of hyaloid vascular regression during mouse neonatal eye development and the first three-dimensional images of the complex hyaloid vascular structure.

The retrolental vitreous in the newborn mouse eye contains the hyaloid vascular system, composed of intraocular vessels that nourish the immature lens. ¹ The hyaloid artery (HA) arises from the main ophthalmic artery as a single trunk and enters the developing eye through the embryonic fissure. ² In its passage across the vitreous cavity, the artery ramifies into a large number of branches: the vasa hyaloidea propria (VHP). ³ As the HA reaches the posterior pole of the lens, it divides into many branches that form an anastomotic network over the posterior aspect of the lens: the tunica vasculosa lentis (TVL). ³ In humans, these vessel systems normally regress before birth, whereas in many species of mammals they remain for a certain period after birth. ¹  

The hyaloid system and developing retinal vasculature of the neonatal mouse provide a useful model to investigate physiologically relevant angiogenesis and vascular remodeling. ⁴ During the first two postnatal weeks, the murine intraretinal vasculature develops through vasculogenesis and angiogenic outgrowth from preexisting vessels in response to increasing metabolic demands as neural growth and differentiation proceed. ⁴ Coincident with maturation of the retina and lens, the hyaloid vascular system in the vitreous progressively undergoes vasoregressive events involving capillary lumen occlusion, endothelial cell and pericyte apoptosis, and phagocytic removal by macrophages. ^{1 4} Developmental factors such as increasing ocular size and retinal angiogenesis have been regarded as the background and triggering factors of the regression. ⁵ Changes in the hyaloid may be the consequence of the hydrostatic coupling that exists between this atrophic vasculature and the developing retinal vasculature. ^{6 7}  

Previous light and electron microscopic studies have catalogued the morphology of the regressing hyaloid vascular system in human, rat, rabbit and dog. ⁸ Several hypothetical mechanisms of the vascular regression have been proposed, based on the change of blood flow distribution and vascular obstruction, ¹ although no functional studies have been performed that focused on the blood flow within the regressing ocular vessels. Hemodynamic forces resulting from blood flow direction and rate play an important role in the growth and structure of blood vessels ⁹ and regulate gene expression. ¹⁰ Lack of hemodynamic forces and irregular flow conditions trigger apoptotic degeneration of vascular endothelium by induction of a mechanosensitive autocrine loop of thrombospondin-1 and the αVβ ³ integrin/integrin-associated protein receptor complex. ^{9 11} In capillary segments, flow stasis can result when dying cells project into the capillary lumen and trap blood cells. ⁷  

Vascular and ocular blood flow changes play important pathogenic roles in several ophthalmic diseases, such as diabetic retinopathy and glaucoma. ¹² Failed regression of the hyaloid vascular system underlies an eye disease known as persistent hyperplastic primary vitreous (PHPV), in which the hyaloid vascular system does not atrophy but persists after birth, either patent or atresic. ⁸ This can lead to retinal detachment, cataracts, glaucoma, and retinal degeneration. ¹³ High-frequency ultrasonography is a noninvasive method of in vivo evaluation of the eye at high resolution that can be reliably used to distinguish characteristic features of human PHPV ¹⁴ and that we have used to characterize mouse embryonic and postnatal ocular development. ^{15 16} Doppler ultrasound involves measurement of the frequency shift resulting from acoustic scattering by moving blood particles. Extending Doppler imaging to the high-frequency domain (20–55 MHz) allows detection and characterization of blood flow in small ocular vessels with high spatial resolution. ¹⁷  

We used high-frequency ultrasound and microcomputed tomography (microCT) to characterize hyaloid vascular regression in neonatal CD-1 mice. The hyaloid vasculature provides a unique opportunity to observe the functional interaction of apoptotic and angiogenic development in a well-regulated context. In this article, we present the first noninvasive in vivo study of the functional impact of the dynamic process of hyaloid vascular regression from P0 to P16, including blood flow velocity measurements in the retina, HA, VHP, and TVL. Three-dimensional (3-D) microCT is also used to provide intralumenal images of hyaloid vascular structure. The present study outlines imaging techniques that could assist in future studies in examining the mechanism of vascular regression. The microimaging techniques used in this study could be applied to the study of various vascular beds in normal murine development and mouse models of human disease. 

Materials and Methods

Ultrasound Biomicroscopic Imaging and Blood Flow Velocity Measurements

Ultrasound biomicroscopic (UBM) imaging was performed (VS40 ultrasound biomicroscope; VisualSonics, Toronto, Ontario, Canada) with the system operating at a center frequency of 40 MHz with B-scan imaging and Doppler flow measurement capabilities, as described previously. ^{15 16} The UBM system provided 40-μm axial by 60-μm lateral resolution in an 8 × 8-mm image plane at a 4-Hz frame rate. All animal experimentation was performed under an approved animal care protocol in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eyes from CD-1 mice (Charles River, St. Constant, Quebec, Canada) were examined from the day of birth to 16 days of postnatal development. Mice were anesthetized with ketamine-xylazine (10 mg/g body weight and 2 mg/g body weight, respectively) and imaged on a mouse imaging stage (THM100; Indus Instruments, Houston, TX). Imaging was performed with ultrasound gel as a coupling fluid on the skin (before eyelid opening) or corneal surface (after eyelid opening). Care was taken to place the subjects in similar postures to ensure similar orientation of vessels during imaging. Retinal blood flow was measured one optic nerve head diameter from the center of the optic nerve. At the TVL, lens blood flow was measured directly above the central HA and at the median point between the central HA and the lateral boundary of the lens major axis on either side of the lens (for measurement positions, see Fig. 1 ). HA blood flow was measured at the median point between the junction of the HA with the retina and the junction with the lens. Doppler sample volume placement for VHP blood velocity measurement was selected on the first branch of the VHP superior to the retina, at the median point between the junction of the VHP with the HA and the junction of the VHP with the lens. Some animals were euthanatized by carbon dioxide asphyxiation after imaging and their heads fixed in 10% formalin for histologic sectioning and hematoxylin and eosin staining performed by the Histopathology Core of the Centres for Modeling Human Disease (Toronto, Ontario, Canada). Statistical analysis was performed with one-way analysis of variance (ANOVA; Origin ver. 6.1 software; OriginLab Corp., Northampton, MA), as well as nonparametric ANOVA (Prism4 software; GraphPad Software Inc., San Diego, CA), since the small population studied may have invalidated the assumption of normality for ANOVA testing. Statistical significance was determined as P < 0.05. 

MicroCT Imaging

MicroCT scanning was performed (MS-8 microCT scanner; Enhanced Vision Systems, Inc., London, Ontario, Canada), using the same scanning protocol for each scan (4 hours, 80 KeV, 90 mA). In preparation for microCT scanning, mice were anesthetized with an intraperitoneal injection of ketamine-xylazine (10 mg/g body weight and 2 mg/g body weight, respectively). CD-1 mice were perfused on postnatal day (P)1, P3, P4, P5, P7, P8, P9, P10, P11, P13, and P16, with a minimum of three animals perfused per time point. The mice were subjected to intracardiac perfusion with a saline flush to remove blood and then were perfused with a silicone rubber contrast agent (Microfil compound MV-122; Flow-Tech Inc., Carver, MA). After dissection, the specimens were stored in 10% neutral buffered formalin at 4°C. Before they were scanned, the specimens were mounted in 10% porcine gelatin (Sigma-Aldrich, Oakville, Ontario, Canada) to minimize motion artifacts. 

Results

Imaging the Hyaloid Vascular Structure with UBM at 40 MHz

UBM was suitable to examine the spatial arrangements of intraocular structures and vessels. UBM B-scan images of murine eyes taken daily from P1 to P16 showed considerable remodeling of the hyaloid vasculature during this period, manifested by progressive reduction in the number of branches of the VHP and decreasing length of the HA. The hyaloid vasculature was extensive at P1 and was progressively reduced in vessel density and complexity (Fig. 2) . The greatest change in hyaloid vascular structure occurred between P4 and P5, with a reduction in VHP branches evident. No significant hyaloid vasculature structure was evident after P12 (Fig. 2) . Histologic sections stained with hematoxylin and eosin provided evidence of the hyaloid vascular structure, but due to the complex, 3-D nature of the hyaloid vascular system structure, the sections were of limited utility. 

Quantification of the Hyaloid Vascular System Blood Velocity

In wild-type CD-1 neonates, blood flow was detected in the HA, VHP, and TVL with high-frequency UBM. Sample volumes for blood flow velocity measurement were selected as illustrated in Figure 3 . Analysis of variance indicated a difference in mean peak blood flow velocity in the HA, with significant increase observed from P3 to P4, then significant decreases between P6 and P7 and between P11 and P12 (ANOVA; all P < 0.01). Nonparametric ANOVA of HA flow data indicated significant changes between P2 and P4 (P < 0.01), P4 and P7 (P < 0.001), P5 and P8, and P6 and P8, as well as between several early postnatal time points (P0, P1, P3, P4, P5, and P6) and P10. No further blood flow was detected after P12 (Fig. 4 , Table 1 ). VHP blood flow differed with a trend of decreasing mean peak blood velocity between P1 and P2 and between P8 and P9 (ANOVA; all P < 0.01). Nonparametric ANOVA indicated that the most significant decrease in VHP flow occurred at P9, with significant decreases from multiple early time points (P0, P1, P2, P3, P4, P5, P6, and P8) and P9, as well as from P1 to P7. An inverse trend of increasing mean peak retinal blood flow velocity and decreasing mean peak lens blood flow was also observed (Fig. 5 , Table 2 ). Nonparametric ANOVA indicated that the most significant increase in retinal flow occurred at P8, with significant increases between early time points (P0, P1, P2, and P3) and P8 (all P < 0.001), and no significant changes in lens flow determined by nonparametric ANOVA. 

Imaging of Vascular Changes

MicroCT was used to produce 3-D images of the mouse hyaloid vasculature at 10.85-μm resolution. Detailed images of hyaloid vascular architecture were obtained through its regression, from a highly branched structure at P0 (Fig. 6A)with highly visible TVL surrounding the lens (Figs. 6B 6C)to the absence of hyaloid vessels at P16 (Fig. 6F) . Retinal vessels were evident at P13 (Fig. 6E)and P16 (Fig. 6F) . 3-D microCT images of contrast-perfused hyaloid vasculature can be rotated 360° using the software (MicroView; Enhanced Vision Systems, Inc.), permitting examination of the hyaloid vasculature from multiple vantage points, as shown in a 3-D rotating movie clip of P9 mouse ocular vasculature (Movie 1). Additional surrounding vasculature associated with the choroidal circulation and vasculature associated with soft tissue such as the eyelids are visible at the extreme bottom and top of the image, respectively. Some truncations of the vessels are visible, particularly in the anterior ciliary artery seen encircling the anterior segment, due to image-cropping to provide the most informative view of hyaloid vascular structure. 

Discussion

Mouse models are increasingly important in biomedical research, and experimental studies in mice have been useful in understanding disease mechanisms. The development and adult structure of the eye are similar in rodents and primates ¹⁸ ; however, mice present unique optical and photographic challenges due to their small eye size and the small radius of curvature of the cornea and lens. ¹⁹ There is therefore considerable interest in noninvasive imaging modalities for the assessment of ocular blood flow. During hyaloid vascular regression, the lack of enveloping tissue and its easy accessibility in postnatal animals provide an opportunity to evaluate ocular vasculature in vivo using ultrasound imaging, with fine vascular detail more readily imaged by microCT. Individual histologic sections were of limited utility for the visualization of the hyaloid vasculature, although this might be improved by the use of 3-D reconstruction software. 

Despite the large number of studies describing the morphology of intraocular vessels, the cessation of blood flow during developmental regression of the hyaloid vessels has not been studied in a quantitative manner. The HA continues to develop even after birth, whereas other hyaloid vessels begin to regress shortly after birth. ¹ In the VHP, there was little change in the number of vessels on days 0 and 4, but then the VHP rapidly decreased in number between days 5 and 10, as previously reported. ^{1 8 20} In the VHP, regression occurred segmentally and resulted in a decreased number of branches, as reported previously. ¹ This is consistent with reports that apoptosis in the hyaloid vascular system peaks between P7 and P8, ²¹ and obstruction of capillaries occurs more often at stages after P8. ¹ The finding that VHP branches rapidly decreased in number after the age of 8 days may be due to the changed distribution of the blood flow between the VHP and developing retinal arteries. The atrophy of the VHP has been demonstrated to be a consequence of changes in the hydrostatic coupling between the VHP and the developing retinal vasculature. ⁶ In mice, the biphasic development of the retinal vasculature begins with the growth of spokelike peripapillary vessels radially from the central retinal artery and vein by vasculogenesis, becoming progressively interconnected by a superficial capillary plexus between P0 and P10. ^{22 23} The second, angiogenic phase of retinal vessel formation occurs between P8 and P18 when sprouts from capillaries of the superficial vascular layer penetrate the retina, where their tips branch laterally to form intermediate and deep capillary beds. ^{22 23}  

In the hyaloid vascular system of rats, apoptotic regression occurs first in vessels that are hemodynamically disadvantaged and have less blood flow, suggesting that the decrease in blood flow may be one of the major triggering factors of regression in these vessels. ¹ Regressing vessels observed are characterized by apoptosis or obstruction with or without narrowing of the lumen. ¹ Obstruction may occur first in the intact lumen, and the consequent cessation of blood flow may trigger segment narrowing; or the obstruction may occur at the luminal portion narrowed by endothelial degeneration, and the cessation of blood flow may cause further obstruction of the vascular segment. ¹ It has also been shown that the occurrence of apoptosis in the pupillary membrane correlates to flow status: As the flow decreased, the appearance of apoptosis in capillaries increased, ²⁴ and the same correlation is thought to exist in the hyaloid vascular system. As a key role of the VHP is thought to be nourishment of the retina before the maturation of retinal vessels, the VHP should regress after the completion of retinal vessels. ¹ By what mechanism the distribution of blood flow is changed and the segmental regression of capillaries is induced by decreased blood flow remain to be studied. Smooth muscle cells in the tunica media at the proximal portion of the HA may be playing some regulatory role in the distribution change. ¹  

The mouse hyaloid vascular system normally regresses during the first 2 weeks of postnatal development, although time courses for changes in the number of vessels in the VHP, TVL, and HA can vary slightly from strain to strain. ^{1 21} Some variability in the peak blood flow velocities in this study were evident from the standard deviations, and part of this variability may be due to the natural variation in the time course of hyaloid vascular system regression. Macrophages are required for and are actively involved in the regression of the hyaloid vascular system, through phagocytosis of the cellular debris resulting from tissue regression and occlusion of the capillary lumen of hyaloid vessels, but are more numerous in some eyes than in others. ^{8 20} This would be likely to cause some variability in the physical regression of the hyaloid vessels. The transition from retinal vasculogenesis to angiogenesis from P8 to P10 may explain some of the intersubject variation in retinal peak blood velocities we observed at these time points. Variations in the Doppler measurements may also be partly attributable to slight differences in interrogation locations in the blood vessels and differing incident angles. Measurements were made in mice with similar heart rates to ensure a representative level of hemodynamic stability of the animals when quantitating blood flow. In rabbits, the injection of anesthetics can induce a temporary local contraction of the vessel wall that lasts until mixing of the anesthetic and the blood is achieved (i.e., with a time scale of a few seconds). ²⁵ We used intraperitoneal administration of the anesthesia, which probably would not have created the same degree of vessel wall contraction as intravenous bolus administration. 

We have quantitated the diminishing mean blood flow velocities in the period during which the hyaloid vascular system is known to undergo regression. The mouse eye appears to be a good model for human hyaloid vascular regression. Peak velocities observed in the mouse were similar to those in the human HA, which have been reported to be approximately 4 cm/sec. ²⁶ We observed an anterior-to-posterior pattern of HA regression, consistent with previous observations in humans, ²⁷ although the precise mechanisms of regression of hyaloid vessels remain for further studies. Pathologic features of ocular diseases such as diabetic retinopathy can include vascular atrophy ²⁸ ; however, any similarity to the sequence of events in hyaloid vascular regression is unclear. Ultrasound biomicroscopy and microCT may be useful experimental tools in future studies of mouse ocular development and mouse models of ocular disease. 

 Supported by the Ontario Research and Development Challenge Fund through the Ontario Small Animal Imaging Consortium.

 Submitted for publication July 20, 2004; revised November 5 and December 13, 2004, and January 19, 2005; accepted February 2, 2005.

 Disclosure: A.S. Brown, VisualSonics, Inc. (C); L. Leamen, None; V. Cucevic, None; F.S. Foster, VisualSonics, Inc. (C, F, I, P)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Schematic of the mouse hyaloid vascular system, indicating locations of Doppler sample volume placement (rectangular box) for measurements taken of the retina, TVL, HA, and VHP.

Figure 2.

 

Images of mouse eye shows highly branched vasculature extending from the retina to the posterior lens surface at P0 (A), P4 (B), and P7 (C). Several branches had regressed, and fewer vessels were visible in the vitreous cavity at P9 (D) and P13 (E), with no vessels evident at P16 (F).

Figure 3.

 

Sample B mode image of mouse eye containing hyaloid vasculature at P3 (A) and an example of Doppler sample volume placement for velocity measurements with 8 × 8-mm and 3 × 3-mm fields of view (B). A representative Doppler blood flow waveform for mouse HA at P3 is shown in (C).

Figure 4.

 

Mean blood flow velocity in the mouse HA and VHP from P0 to P16. Error bars, SD.

Table 1.

 

Calculated HA and VHP Peak Blood Flow Velocities

Age	HA Peak Blood Flow Velocity					VHP Peak Blood Flow Velocity
		Mean (cm/s)	SD	n	Mean (cm/s)	SD	n
P0	2.98	0.95	5	1.43	0.36	5
P1	3.20	1.18	7	1.89	0.33	7
P2	2.93	1.32	6	1.06	0.39	6
P3	3.10	0.63	6	0.54	0.40	6
P4	3.82	1.18	5	1.25	0.40	5
P5	4.18	1.69	5	1.29	0.59	5
P6	3.56	1.29	5	1.21	0.52	5
P7	2.58	0.87	6	0.86	0.54	6
P8	2.25	1.14	7	0.98	0.46	7
P9	0.93	0.90	5	0.16	0.16	5
P10	1.84	0.45	5	0.66	0.30	5
P11	2.25	0.89	6	0.57	0.06	6
P12	1.35	0.78	5	0.43	0.33	5
P13	0	0	6	0	0	6
P14	0	0	5	0	0	5
P15	0	0	5	0	0	5
P16	0	0	6	0	0	6

 Data are those shown in Figure 4(mean ± SD). n, number of samples measured.

Figure 5.

 

Mean blood flow velocity in the mouse retina and lens from P0 to P12. Increasing retinal vascular blood flow was observed as lens blood flow velocity diminished. Error bars, SD.

Table 2.

 

Calculated Retinal and Lens Peak Blood Flow Velocities

Age	Retinal Peak Blood Flow Velocity					Lens Peak Blood Flow Velocity
		Mean (cm/s)	SD	n	Mean (cm/s)	SD	n
P0	0.32	0.09	5	1.08	0.25	5
P1	0.71	0.29	7	1.20	0.42	7
P2	1.22	0.29	6	0.81	0.18	6
P3	0.58	0.07	6	0.68	0.19	6
P4	4.62	1.09	5	0.55	0.07	5
P5	2.70	0.94	5	0.89	0.20	5
P6	5.26	1.79	5	0.90	0.22	5
P7	4.30	0.07	6	0.14	0.22	6
P8	3.53	1.73	7	0.67	0.38	7
P9	ND	ND	ND	0.19	0.19	5
P10	2.79	0.14	5	0.19	0.19	5
P11	ND	ND	ND	ND	ND	ND
P12	4.35	0.52	5	0.24	0.28	5

 Data are those shown in Figure 5 . n, number of samples measured. Time points not evaluated are indicated by ND.

Figure 6.

 

MicroCT images of a mouse eye at P0 (A) show the HA (arrow) and extensive branching through the vitreous (VHP) and across the posterior surface of the lens (TVL), also visible at P4 (B), P7 (C) and P9 (D). The hyaloid vasculature was no longer detected at P13 (E) and at P16 (F), although the retinal vasculature was evident. Scale bars, 500 μm.

Supplementary Materials

Movie 1 - 1.33 MB