Figure 4.

Genetic deletion of A₁Rs reduced hypoxia-induced physiological intraretinal revascularization without affecting intravitreal neovascularization, hypoxia-induced cellular apoptosis, and proliferation at P17. (A) Retinal vasculature was stained by isolectin B4 of whole-mount retinas at P17 of OIR. Whole retinal area was circumscribed by white dotted line (n = 13 retinas from 13 WT mice and n = 12 retinas from 12 A₁R KO mice). Avascular area and the areas of neovascularization tufts were highlighted in yellow and green, respectively. Scale bar: 500 μm. (B) The avascular area (%) was quantified as the ratio of central avascular area to whole retinal area. The neovascularization tufts area (%) was quantified as a percentage of whole retinal area. (C) Retinal pathologic angiogenesis at P17 was observed by using hematoxylin and eosin staining (n = 7 retinas from 7 WT mice and n = 7 retinas from 12 A₁R KO mice). Nuclei on the vitreal side of the inner limiting membrane are indicated by black arrows. Scale bar: 50 μm. (D) The number of neovascular nuclei was quantified. (E) Hypoxia-induced retinal cellular proliferation of WT and A₁R KO mice at P17 of OIR was detected by PCNA immunohistochemistry (n = 7 retinas from 7 WT mice and n = 7 retinas from 12 A₁R KO mice). The PCNA-positive cells are indicated by yellow arrows. Scale bar: 20 μm. (F) The quantification of PCNA-positive cells of WT and A₁R KO mice is shown. (G, H) Hypoxia-induced apoptotic cells of WT and A₁R KO retinas at P17 of OIR were assayed by TUNEL staining and costaining with CD31. TUNEL-positive cells are indicated by yellow arrows (n = 14 retinas from 14 WT mice and n = 15 retinas from 15 A₁R KO mice). Coimmunostaining of TUNEL and CD31⁺ cells revealed that CD31⁺ cells (white arrowhead) were largely segregated from the TUNEL signal (yellow arrowhead) with little coimmunostaining of TUNEL/CD31 signal (yellow arrow). Scale bar: 20 μm. (I) The quantification of TUNEL-positive cells of WT and A₁R KO mice is shown. Data in (B, D, F, I) are presented as mean ± SEM. ***P < 0.001 (Student's t-test), comparing A₁R KO group with WT group.