Platelet-Derived Growth Factor Over-Expression in Retinal Progenitors Results in Abnormal Retinal Vessel Formation

<div><p>Platelet-derived growth factor (PDGF) plays an important role in development of the central nervous system, including the retina. Excessive PDGF signaling is associated with proliferative retinal disorders. We reported previously that transgenic mice in which PDGF-B was over-expressed under control of the nestin enhancer, nes/tk-PdgfB-lacZ, exhibited enhanced apoptosis in the developing corpus striatum. These animals display enlarged lateral ventricles after birth as well as behavioral aberrations as adults. Here, we report that in contrast to the relatively mild central nervous system phenotype, development of the retina is severely disturbed in nes/tk-PdgfB-lacZ mice. </p> <p>In transgenic retinas all nuclear layers were disorganized and photoreceptor segments failed to develop properly. Since astrocyte precursor cells did not populate the retina, retinal vascular progenitors could not form a network of vessels. With time, randomly distributed vessels resembling capillaries formed, but there were no large trunk vessels and the intraocular pressure was reduced. In addition, we observed a delayed regression of the hyaloid vasculature. The prolonged presence of this structure may contribute to the other abnormalities observed in the retina, including the defective lamination.</p> </div

Expression of the transgene in the developing retina.

<p>X-gal staining in sectioned eyes from transgenic mice at different postnatal days (P1–P30). Pigmented epithelium (pe), neuroblastic layer (nbl), putative ganglion cell layer (p-gcl), inner nuclear layer (inl). Arrowheads in H depict X-gal staining in the inl at P20. Scale bar A–C 200 µm, D–I 50 µm.</p

Partial restoration of Pax2+ and CD31+ migration after STI571 treatment.

<p>Retina flat-mounts of postnatal day 1 (P1) or P2 mice whose mother received the tyrosine kinase inhibitor STI571 (Glivec) from E17.5 to birth (B, B′), or from E17.5 to birth and at P1 (C, C′) and P2 control mice (A, A′). In panels D–G, the inhibitor was administrated to wild-type (D, D′, E, E′) or transgenic (F, F′, G, G′) mice between P7 to P14. Cross sections of retina were stained with antibodies to CD31 and NG2. Outer nuclear layer (onl), inner nuclear layer (inl), ganglion cell layer (gcl). Scale bar A–C, A′–C′ 200 µm, D–G, D′–G′ 100 µm.</p

Glial activation in the PDGF-B transgenic retina.

<p>Retinas from wild-type (A, C) and transgenic (B, D) mice were stained for Pax2 on postnatal day 1 (P1) and P5 to depict astrocyte precursors. Mature astrocytes were visualized with GFAP antibodies on retinal flat-mounts from wild-type (E) and transgenic mice (F). Panels G and H are magnifications of E and F. The insert in H shows astrocyte morphology in the transgenic retina (the same cells are indicated by an arrow). Scale bar A–D 200 µm, insets 100 µm, E–F 1 mm, G–H 100 µm.</p

Transgenic mice exhibit reduced intraocular pressure.

<p>Measurement with a rodent tonometer revealed significantly lower pressure in the eyes of transgenic (TG) than in wild-type (WT) animals. The arrow indicates the reference value for C57Bl/6 mice.</p

Morphological alterations in eyes of mice over-expressing PDGF-B.

<p>Eyes from wild type (A) and transgenic (B) mice on postnatal day 15 (P15) were photographed after dissection. Folding was observed in the transgenic (D) retina, compare to wild type (C). The eye-pairs showed in (A) and (B) are from the same individual, respectively, and the retinas shown in (C) and (D) were dissected from the eyes shown in (A) and (B, large eye). Histological examination (DAPI nuclear stain) revealed a disorganization of retinal lamination (F) not observed in wild type eyes (E). Outer nuclear layer (onl), inner nuclear layer (inl), ganglion cell layer (gcl), Scale bar 50 µm.</p

Failure to vascularize the retina.

<p>Staining of cross-sections and flat-mounts with antibodies against CD31, NG2 and GFAP on postnatal day 5 (P5, (A–F, M–O) and P60 (G–L). The white arrowhead in A and C indicates the border of CD31-positive cells in a control mouse. Note their absence in the transgenic retina (B and D). NG2, CD31 and GFAP staining is localized to the retrolental cell mass, indicative of the hyaloid, in transgenic retinas (D, F, M–O) compared to wild-type retinas (C, E). Irregular CD31+ cells had developed at P60 in the transgenic retina (H, J, L). Wild-type control (G, I, K). M–O: The same P5 transgenic retina as shown in D, stained for NG2 (red) and CD31 (green) and DAPI (blue). Tractional forces on the retina have caused it to fold (thin white arrow in M) by sprouting ectopic and irregular blood vessels (black arrows in N) that infiltrates the retina at various depths. Outer nuclear layer (onl), inner nuclear layer (inl), ganglion cell layer (gcl), optic nerve exit (one). Scale bar A and B 1 µm, C–F 200 µm, G–H 1 mm, I–J 200 µm, K–L 50 µm and M 200 µm.</p

Analysis of makers for photoreceptors and horizontal cells.

<p>Wild-type (A, C, E, G, I, K) and transgenic (B, D, F, H, J, L) retinas were cross-sectioned and stained with the antibodies indicated at ages P5, P30 and P60. Scale bar A–L 50 µm. Outer nuclear layer (onl), inner nuclear layer (inl), ganglion cell layer (gcl).</p

Number, retinal area and Brn3a⁺RGC density in experimental flat-mount retinas 7 days post-lesion.

<p>Number, retinal area and Brn3a⁺RGC density in experimental flat-mount retinas 7 days post-lesion.</p

Number, retinal area and Brn3a+ RGC density in embryonic and post-natal control retinas.

<p>Number, retinal area and Brn3a+ RGC density in embryonic and post-natal control retinas.</p