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Diana C Lozano, Hailey Pausz, William O. Cepurna, Virginia O'Callahan, Susan Wentzien, Katherine Delf, Shandiz Tehrani, Elaine C. Johnson, John C Morrison; Time Course of Optic Nerve Head (ONH) Glia Proliferation following a Pulse-Train Controlled Elevation of IOP (PT-CEI). Invest. Ophthalmol. Vis. Sci. 2020;61(7):260.
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In our chronic glaucoma model, we recently reported that the majority of ONH proliferating cells were astrocytes. However, we were unable to unmask the time course of this response. Here, we utilized our PT-CEI model, for which we know the duration and level of IOP elevation, and quantified proliferating glia immediately after (0Hr) and up to 1 month following PT-CEI.
The anterior chamber of anesthetized Brown Norway rats was cannulated and exposed unilaterally to 60 mmHg for 8 hours, with IOP normalization (20 mmHg) for 5 minutes every hour. Animals were euthanized 0Hr, 1, 2, 3, 7, 10 days or 1 month after PT-CEI (4–8 animals per time point). ONH longitudinal sections were co-immunolabeled with nuclear markers Ki67 (proliferative nuclei) and either Sox2 (astrocytic marker) or Iba1 (microglia/macrophage marker) and DAPI to identify ONH nuclei. Cell densities were quantified in the anterior (first 150µm) and transition ONH (250–400µm posterior to BM). One-way ANOVA followed by Dunnett’s multiple comparisons tests were performed to identify nuclear density differences and proportions relative to controls across time points. Nerve cross sections were graded for axonal injury on a scale from 1 (normal) to 5 (majority of axons degenerating).
Small focal optic nerve injury was seen 7–10 days after PT-CEI (grade 1.3±0.3). In control ONHs, 75% of anterior nuclei labeled as Sox2+, while 4% were Iba1+. In the transition region, proportions were 47% Sox2+ and 7% Iba1+. Up to 1 month after PT-CEI, total nuclear and astrocyte densities remained unchanged. Iba1+ cells significantly increased in both regions at 7 days (P<.001, both). Anteriorly, proliferating cell density significantly increased at 1–7 days (P<0.001). By 2 days, 19% of all anterior nuclei were Ki67+ and 52% of these co-labeled with Sox2+. In the transition region, 10% of all nuclei were Ki67+ by 7 days (P < .0001) and 76% of these co-labeled with Sox2+. By 1 month, <1% of nuclei in both regions were Ki67+.
This study identifies proliferation as an early event that resolves one month after PT-CEI. As in the chronic model, and as suggested by our RNA-seq reports (ARVO E-abstracts 2536, 3740, and 675), ONH cell proliferation is a prominent and early response to elevated IOP exposure.
This is a 2020 ARVO Annual Meeting abstract.
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