Figure 1
Figure 1. Sema3A expression is consistent with a role in retinopathy. (A) Frozen cross-section (left panel) and flat-mount retinas (central panel) taken at P17 of OIR demonstrating the principal characteristics of PRs including avascular (A) and vascular (V) zones. Paraffin sections (right panel) demonstrating pre-retinal neovascular tufts (black arrows). (B) Real-time PCR on whole retinas taken at P8 and P14 demonstrates an ∼ 3-fold increase in Sema3A during the vaso-obliterative and neovascular phases of PRs, respectively (n = 3). Values are gene copy number normalized to CyclophilinA standards ± SEM. **P = .0015 and *P = .0157 compared with normoxia (Norm). (C) Microdissection of avascular regions of the OIR retina at P14 reveals a 3.5-fold induction in Sema3A protein levels in the avascular area (n = 3). Values are shown relative to vascularized areas ± SEM. *P = .0447 compared with the vascularized zone (V). At P19, when physiologic retinal revascularization is reinstated, Sema3A in the avascular retina returns to control levels. Levels of Sema3A in the peripheral (P) and central (C) retina of normoxic controls are comparable. (D) Laser capture microdissection on retinal layers (F) (also see supplemental Figure 1B) demonstrates that Sema3A is primarily produced in the ganglion cell layer (GCL), with lower expression in the inner nuclear layer (INL). ONL indicates outer nuclear layer. Levels of Sema3A surge 4.4-fold in the GCL at P14 after OIR (n = 3). **P = .0075 and *P = .0143 relative to normoxia (Norm). (E) Confocal imaging of immunohistochemistry on central avascular retinal cross-sections (OIR P14) reveals a predominant expression of Sema3A by RGCs as confirmed by merging with RGC marker Thy1.1. (G) At P17, laser capture microdissection and RT-PCR of normal vessels versus neovascular tufts revealed a 2.2-fold induction in Nrp-1 in tufts (n = 3). Values are gene copy number normalized to CyclophilinA standards ± SEM. ***P = .0007 compared with controls (Ctl). (H) Immunohistochemistry on flat-mount retinas confirms pronounced staining of Nrp-1 on lectin-stained neovascular tufts (P17). Images are representative of 5 experiments. Scale bars represent 300 μm (A right panel), 500 μm (A central panel), 50 μm (A right panel) 25 μm (E), 100 μm (F), and 25 μm (H).

Sema3A expression is consistent with a role in retinopathy. (A) Frozen cross-section (left panel) and flat-mount retinas (central panel) taken at P17 of OIR demonstrating the principal characteristics of PRs including avascular (A) and vascular (V) zones. Paraffin sections (right panel) demonstrating pre-retinal neovascular tufts (black arrows). (B) Real-time PCR on whole retinas taken at P8 and P14 demonstrates an ∼ 3-fold increase in Sema3A during the vaso-obliterative and neovascular phases of PRs, respectively (n = 3). Values are gene copy number normalized to CyclophilinA standards ± SEM. **P = .0015 and *P = .0157 compared with normoxia (Norm). (C) Microdissection of avascular regions of the OIR retina at P14 reveals a 3.5-fold induction in Sema3A protein levels in the avascular area (n = 3). Values are shown relative to vascularized areas ± SEM. *P = .0447 compared with the vascularized zone (V). At P19, when physiologic retinal revascularization is reinstated, Sema3A in the avascular retina returns to control levels. Levels of Sema3A in the peripheral (P) and central (C) retina of normoxic controls are comparable. (D) Laser capture microdissection on retinal layers (F) (also see supplemental Figure 1B) demonstrates that Sema3A is primarily produced in the ganglion cell layer (GCL), with lower expression in the inner nuclear layer (INL). ONL indicates outer nuclear layer. Levels of Sema3A surge 4.4-fold in the GCL at P14 after OIR (n = 3). **P = .0075 and *P = .0143 relative to normoxia (Norm). (E) Confocal imaging of immunohistochemistry on central avascular retinal cross-sections (OIR P14) reveals a predominant expression of Sema3A by RGCs as confirmed by merging with RGC marker Thy1.1. (G) At P17, laser capture microdissection and RT-PCR of normal vessels versus neovascular tufts revealed a 2.2-fold induction in Nrp-1 in tufts (n = 3). Values are gene copy number normalized to CyclophilinA standards ± SEM. ***P = .0007 compared with controls (Ctl). (H) Immunohistochemistry on flat-mount retinas confirms pronounced staining of Nrp-1 on lectin-stained neovascular tufts (P17). Images are representative of 5 experiments. Scale bars represent 300 μm (A right panel), 500 μm (A central panel), 50 μm (A right panel) 25 μm (E), 100 μm (F), and 25 μm (H).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal