Figure 4

Minocycline (Mino) treatment significantly diminishes cellular inflammation following ischemia-reperfusion (IR). Mino was delivered as twice-daily intraperitoneal (IP) injections, with two initial dosages of 45 mg/kg prior to ischemia and dosages of 22.5 mg/kg just prior to ischemia and every 12 h for the next 2 d during the reperfusion period. Non-treated (NT) animals received PBS vehicle injections. One eye of each rat was subjected to IR for 45 min or needle puncture only (Sham) and after 48 h of reperfusion retinas were enzymatically dissociated and cells analyzed by flow cytometry. (A) Representative scatter plots of CD11b and CD45 immunostaining intensities of total retinal cells from Sham and IR retinas of rats treated with PBS or Mino. Events are partitioned into quadrant 1 (Q1) containing CD11b⁺/CD45^low cells, quadrant 2 (Q2) containing CD11b⁺/CD45^hi cells and quadrant 3 (Q3) containing CD11b^neg/CD45^hi cells. The fourth quadrant contains the majority of retinal cells, which are CD11b^neg/CD45^neg. (B) Example of MHCII staining distributions of cells from quadrants 1, 2 and 3. Red traces are for cells from Sham-treated retinas, blue traces are for cells from IR retinas and the black trace is for cells incubated without the MHCII antibody and used to define the intensity cutoff between define MHCII⁺ and MHCII^neg cells. (C) Quantification of total events, MHCII⁺ events and MHCII^neg events from quadrants 1, 2 and 3 from Sham and IR retinas from PBS- and Mino-treated rats. The numbers shown are mean percentiles relative to all events, including those in quadrant 4, obtained from three separate flow experiments, with a total of 11 to 12 determinations per group. Significant differences between Sham and IR groups are indicated as *P ≤0.05, **P ≤0.01 and ***P ≤0.001, while significant effects of Mino-treatment are indicated by #P ≤0.05 and ##P ≤0.01. All comparisons are by Students t-test.