Retinal vaso-obliteration of wild type and <i>Fzd4</i>^+/- mice after ocular ischemic retinopathy.

Wild type and Fzd4+/- mice were exposed to our OIR model from P7 to P12. (A) At P12 whole mounted retinas were stained with Alexa Fluor 594-conjugated GS-IB4 lectin and visualized using confocal microscopy (panel i). Vaso-obliterated areas are shown in white (panel ii). (B). Quantification of the vaso-obliterated area. Imaging analyses are expressed as mean ± standard error of the mean (SEM) and P values were calculated using a one-way ANOVA unpaired t-test. NS, not significant.</p

Retinal vasculature development into the deep and intermediate layers of wild type and <i>Fzd4</i>^+/- mice under normoxic conditions.

Whole mount retinas of Fzd4+/+ and Fzd4+/- under normoxic conditions were stained with isolectin-594 and imaged at 25x magnification using a Zeiss LSM 510 Meta laser scanning confocal microscope. Z-stacks representing vascular growth in the deep and intermediate layers at the mid periphery were generated using 1 micron slices. Z-stacks were analyzed using the ImageJ software program. Green bars represent the X-axis, blue bars represent the Y-axis and purple represent the Z-axis. White arrows indicate examples of vessel branching in retinal layers.</p

Delayed revascularization in <i>Fzd4</i>^+/- mice after ocular ischemic retinopathy.

<p>Wild type and <i>Fzd4</i>^+/- mice were exposed to our OIR model from P7 to P12 and returned to room air. At P17 (<b>A</b>) whole mounted retinas were stained with Alexa Fluor 594-conjugated GS-IB4 lectin and visualized using confocal microscopy (panel <i>i</i>), vaso-obliterated areas are shown in white (panel <i>ii</i>). (<b>B</b>). Quantification of the vaso-obliterated area. Imaging analyses are expressed as mean ± standard error of the mean (SEM) and <i>P</i> values were calculated using a one-way ANOVA unpaired t-test. **P<0.01.</p

Decreased vessel number and increased vessel caliber in <i>Fzd4</i>^+/- mice after ocular ischemic retinopathy.

<p>Comparison of vessel number and caliber for wild-type and <i>Fzd4</i>^+/- mice after ocular ischemic retinopathy at P12, P17 and P25. (<b>A</b>) For vessel count, the number of vessels at lines in each quadrant at proximal, mid, and distal regions from the optic nerve was determined. (<b>B</b>) The width of vessels at proximal, mid, and distal regions from the optic nerve was determined was calculated using Image J. A 50 μm cutoff was chosen to represent the width of major primary vessels. Imaging analyses are expressed as mean ± standard error of the mean (SEM) and <i>P</i> values were calculated using a one-way ANOVA unpaired t-test. * P<0.05, **<0.01, ***<0.001. VO, vaso-obliterated. NS, not significant.</p

<i>Fzd4</i> Haploinsufficiency Delays Retinal Revascularization in the Mouse Model of Oxygen Induced Retinopathy

<div><p>Mutations in genes that code for components of the Norrin-FZD4 ligand-receptor complex cause the inherited childhood blinding disorder familial exudative vitreoretinopathy (FEVR). Statistical evidence from studies of patients at risk for the acquired disease retinopathy of prematurity (ROP) suggest that rare polymorphisms in these same genes increase the risk of developing severe ROP, implying that decreased Norrin-FZD4 activity predisposes patients to more severe ROP. To test this hypothesis, we measured the development and recovery of retinopathy in wild type and <i>Fzd4</i> heterozygous mice in the absence or presence of ocular ischemic retinopathy (OIR) treatment. Avascular and total retinal vascular areas and patterning were determined, and vessel number and caliber were quantified. In room air, there was a small delay in retinal vascularization in <i>Fzd4</i> heterozygous mice that resolved as mice reached maturity suggestive of a slight defect in retinal vascular development. Subsequent to OIR treatment there was no difference between wild type and <i>Fzd4</i> heterozygous mice in the vaso-obliterated area following exposure to high oxygen. Importantly, after return of <i>Fzd4</i> heterozygous mice to room air subsequent to OIR treatment, there was a substantial delay in retinal revascularization of the avascular area surrounding the optic nerve, as well as delayed vascularization toward the periphery of the retina. Our study demonstrates that a small decrease in Norrin-Fzd4 dependent retinal vascular development lengthens the period during which complications from OIR could occur.</p></div

Changes in vessel number and calibre under normoxic conditions for wild type and <i>Fzd4</i>^+/- mice.

<p>Mice were raised in room air and at P12, P17, and P25 whole mounted retinas were stained with Alexa Fluor 594-conjugated GS-IB4 lectin and visualized using confocal microscopy. (<b>A</b>) For vessel count, profile plots were created by drawing three lines in each of the four quadrants at three specified locations: adjacent to the optic nerve (proximal), adjacent to the periphery of the retina (distal) and midway between the proximal and distal lines (middle). The number of vessels crossing each line in each quadrant was counted. (<b>B</b>) The width of vessels at proximal, mid, and distal regions from the optic nerve was calculated using Image J. A 50 μm cutoff was chosen to represent the width of major primary vessels and calculated as a percentage of total vasculature. (<b>C</b>) A schematic representation of vessel number and caliber determination. Line segments were drawn perpendicular to retinal quadrants at the proximal, middle and distal regions. Imaging analyses are expressed as mean ± standard error of the mean (SEM) and <i>P</i> values were calculated using a one-way ANOVA unpaired t-test. *P<0.05, **<0.01, ***<0.001, ****<0.0001. NS, not significant.</p

Toward normalization of vascularization of <i>Fzd4</i>^+/- mice after ocular ischemic retinopathy.

<p>Wild type and <i>Fzd4</i>^+/- mice were exposed to our OIR model from P7 to P12 and returned to room air. At P25 whole mounted retinas were stained with Alexa Fluor 594-conjugated GS-IB4 lectin and visualized using confocal microscopy. The yellow square in the image on the left is enlarged in the panel on the right.</p

Retinal vasculature development into the deep and intermediate layers of wild type and <i>Fzd4</i>^+/- mice after ocular ischemic retinopathy.

<p>Whole mount retinas of <i>Fzd4</i>^+/+ and <i>Fzd4</i>^+/- subjected to the ocular ischemic retinopathy protocol were stained with isolectin-594 and imaged at 25x magnification using a Zeiss LSM 510 Meta laser scanning confocal microscope. Z-stacks representing vascular growth in the deep and intermediate layers at the mid periphery were generated using 1 micron slices. Z-stacks were analyzed using the ImageJ software program. Green bars represent the X-axis, blue bars represent the Y-axis and purple represent the Z-axis. White arrows indicate examples of vessel branching in retinal layers.</p

Analysis of retinal vasculature during development for wild type and <i>Fzd4</i>^+/- mice under normoxic conditions.

<p><b>(A)</b> Wild type and <b>(B)</b> <i>Fzd4</i>^+/- mice were raised in room air. At P12, P17, and P25 whole mounted retinas were stained with Alexa Fluor 594-conjugated GS-IB4 lectin and visualized using confocal microscopy. The yellow square in the image on the left is enlarged in the panel on the right.</p