Figure 4. Capacity of dead/dying neuron clearance in microglia/macrophages was impaired in STAT6-KO mice.

WT and STAT6-KO mice were subjected to 60 minutes of tMCAO. Brains were collected 3d after tMCAO. (A) Representative images of NeuN (blue), TUNEL (green), and Iba1 (red) triple-staining. Scale bar: 10 μm. The inset image depicts the hemisphere ipsilateral to stroke, where brain infarction is shown in gray and the area for image analysis is indicated by the box. (B) High-power 3-D image generated from A. White arrows indicate microglia/macrophages that engulfed dead/dying neurons (Iba1⁺NeuN⁺TUNEL⁺). White arrowheads indicate dead/dying neurons that were not engulfed by microglia/macrophages (Iba1^–NeuN⁺TUNEL⁺). Yellow arrows indicate live neurons (Iba1^–NeuN⁺TUNEL^–). Yellow arrowhead indicates a TUNEL^– neuron touched by an Iba1⁺ cell (Iba1⁺NeuN⁺TUNEL^–). Scale bar: 10 μm. (C) Quantification of the total number of Iba1⁺ microglia/macrophages in ischemic areas as indicated in Figure1A. (D) The number of Iba1⁺NeuN⁺ cells in ischemia areas. (E) The number of Iba1⁺NeuN⁺TUNEL⁺ cells (microglia/macrophages with engulfed dead/dying neurons) in ischemic areas. (F) The number of Iba1^–NeuN⁺TUNEL⁺ nonengulfed dead neurons in ischemic areas was quantified. (G) Phagocytic index, the percentage of dead/dying neurons engulfed by microglia/macrophages ([number of Iba1⁺NeuN⁺TUNEL⁺ cells/number of NeuN⁺TUNEL⁺ cells] × 100%), was calculated in WT and STAT6-KO brains. (H) Quantification of Iba1⁺NeuN⁺TUNEL^– cells (microglia/macrophages colocalize with TUNEL^– neurons) in ischemic areas. n = 6 mice per group. **P ≤ 0.01, ***P ≤ 0.001 STAT6-KO vs. WT, Student’s t test.