Figure 1.
Time course representing the morphological changes found in the fundus of NRV2 mice.
Fundus photographs and fluorescein angiography of NRV2 mice from p17 to p35. (A–E) Fundus images show the emergence of depigmented regions at p17 that increase in size and number through p35. (F–J) Fluorescein angiography indicates vascular leakage corresponding to the areas of depigmentation, peaking at p25 and subsiding by p35. n = 10, Representative images are shown. p = postnatal day.
Figure 2.
Vascular leakage in retinal lesions of NRV2 mice during postnatal development.
Fluorescein angiography images taken of NRV2 mice at the early through late phases after fluorescein injection for p25 and p31. (A–C) At p25 NRV2 mice have increased vascular leakage through the late phase of fluorescein angiography, compare B and C. (D–F) By p31, there is reduced vascular leakage relative to p25, that does not increase from the middle to late phase of fluorescein angiography, compare E and F. Early = 1 minute after fluorescein injection, middle = 3–5 minutes after fluorescein injection, late = 10 minutes after fluorescein injection. n = 10, Representative images are shown. p = postnatal day.
Figure 3.
The peak time for depigmentation and vascular leakage in NRV2 mice is at p25.
Developmental time course of depigmentation and vascular leakage assessed by funduscopy and fluorescein angiography in NRV2 mice. (A) Quantitation of the number of depigmented regions observed by fundus imaging. The peak number of depigmented regions occurs at p25, followed by a relative stabilization. (B) Quantitation of the number of fluorescein angiography leakage, measured by the number of leakage regions, also peaks at p25. Each plot on the graph is an average of 10 eyes, p = postnatal day.
Figure 4.
Vascular leakage corresponds to disturbances in the outer plexiform and outer nuclear layers of the retina.
Images showing the representative time course of lesion development in a NRV2 mouse. (A–C) Fundus image taken at p17, showing areas that have slight depigmentation, at the 1 o’clock position of the image (A, left panel). In the same area, vascular leakage can be seen by fluorescein angiography (B, middle panel). Examination by SD-OCT at the region of vascular leakage (red bar in B) reveals a disturbance in the ONL and OPL of the retina (C, right panel). Arrowheads point to the retinal disturbance. (D–F) Fundus image taken at p21, of the same mouse, shows increased depigmentation at the 1 o’clock position of the image (D, left panel). In the same area vascular leakage can be seen by fluorescein angiography (E, middle panel). Examination by SD-OCT at the region of vascular leakage (red bar in E) reveals a disturbance in the ONL and OPL of the retina (F, right panel). Arrowheads point to the retinal disturbance. (G–I) Fundus image of the same mouse taken at p25, the height of leakage in NRV2 mice, shows depigmentation, at the 1 o’clock position of the image (G, left panel). In the same area, vascular leakage is pronounced compared to the earlier time points as seen by fluorescein angiography (H, middle panel). Examination by SD-OCT at the region of vascular leakage (red bar in H) reveals a disturbance in the ONL and OPL of the retina (I, right panel). Arrowheads point to the retinal disturbance. p = postnatal day, GC: ganglion cells; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS/OS: photoreceptor inner segment/outer segments. n = 10, Representative images are shown. Scale bars: 50 µm.
Figure 5.
Neovessels extend from the INL to the RPE in NRV2 mice.
Retinal cross-sections stained by H&E through a neovascular lesion. (A) Cross-section from a C57Bl6 mouse showing the normal architecture of the retinal layers. (B) Higher magnification of (A) focusing on the normal architecture between the ONL and RPE interface. (C) Cross section from an NRV2 mouse at p25, showing a disturbance in the outer retina. (D) Higher magnification of (C) to show that the lesion is at the interface of the ONL and RPE, in the outer segments of the photoreceptors. (E) Retinal cross-section from another NRV2 mouse at p25, capturing the track of a neovessel spanning from the INL to the RPE. (F) Higher magnification of (E) showing the vessel extending through the ONL. Arrowheads in (C) and (E) indicate the lesion areas that are magnified in (D) and (F). GC: ganglion cells; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS/OS: photoreceptor inner segment/outer segments. n = 3, Representative images are shown. Scale bars: 25 µm.
Figure 6.
Neovessels originate from the INL and grow towards the RPE in NRV2 mice.
Retinal cross-sections stained with isolectin B4 that have been reconstructed into 3D images (Amira software) using confocal Z stacks to show the development of retinal blood vessels in NRV2 mice. (A) Low magnification overview of the 3D reconstruction of a retinal flatmount stained with isolectin B4 for blood vessels. (B–F) High magnification images of retinal cross-sections from NRV2 mice stained with isolectin B4 that have been reconstructed into a 3D montage. (B) At p12, there are only vessels in the INL with no apparent irregular vessel development. (C) At p15, vessels are found extending from the INL towards the RPE cell layer. (D) At p17, the vessels have reached the RPE interface. (E) At p21, vessels have mostly interfaced with the RPE cell layer and have begun to expand across the surface of the RPE. (F) At p25, the vessels have fully interacted with the RPE and formed balloon like structures. p = postnatal day, INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; IS/OS: photoreceptor inner segment/outer segments. The upper black square shows the border of the OPL and ONL. The lower black square shows the interface of the RPE layer. n = 3/timepoint, Representative images are shown. Scale bars: (A): 500 µm, (B–F): 50 µm.
Figure 7.
Neovessels that interface with the RPE form characteristic morphological features of more mature vessels.
Electron micrographs taken of NRV2 mice illustrate commonly observed morphological findings of neovessels when they come in contact with the RPE/Bruch’s membrane. (A) Electron micrograph of a C57BL/6 mouse at 3 months of age shows the normal architecture of the outer retina. (B) Neovessels (outlined in black) from an NRV2 mouse at p21, surrounded by disorganized outer photoreceptor segments. (C) High magnification image (red box in B) that shows the presence of pericytes in the neovessels. (D) A 3 months old, NRV2 mouse with an RPE cell (outlined in black) surrounding the neovessel. (E) High magnification (red box in D) that shows a contact point between the neovessel and RPE cell. (F) Higher magnification (red box in E) of the contact point reveals fenestrations (arrowheads). (G) A 3 months old, NRV2 mouse where the neovessel has come in direct contact with the Bruch’s membrane. IS: inner segment; OS: outer segment; MV: microvilli; BM: Bruch’s membrane; CC: choriocapillaris; NV: new vessels; P: pericyte; n = 3/timepoint, Representative images are shown. Scale bars: (A–D, G): 5 µm, (E, F): 1 µm.