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. 2018 May 22;9:1108. doi: 10.3389/fimmu.2018.01108

Figure 4.

Figure 4

Macrophage subpopulation-specific increases in markers of lysosome activity after experimental stroke. (A) Analysis of transcripts of genes associated with lysosome formation show that lysosomal-associated membrane protein 2 (Lamp2) expression is upregulated after stroke, whereas Lamp1 remains relatively unchanged (sham n = 2, 1 day and 2 days n = 3, 5 days n = 4). (B) RT-qPCR for Lamp2 confirmed significant upregulation of expression 2 days after middle cerebral artery occlusion (MCAO) in comparison to sham-operated animals (naïve n = 4, sham n = 4, 1 day n = 4, 2 days n = 9, 5 days n = 5, 7 days n = 7). (C) Immunofluorescent labeling of LAMP2 (green, LAMP2) along with labeling for both tissue resident and monocyte-derived RPM (blue, F4/80; top panel) marginal zone macrophages (magenta, MARCO; top panel), marginal zone metallophillic macrophages (red, MOMA-1; middle panel), monocyte-derived RPM only (blue, CD11b; middle panel), or tingible body macrophages (red, MOMA-2 within the WP; bottom panel) in spleens from naïve mice, or mice recovered 1–7 days after MCAO or sham surgery (naïve n = 4, sham n = 4, 1 day n = 4, 2 days n = 9, 5 days n = 5, 7 days n = 7). Quantification of co-localization of LAMP-2 immunolabelling with macrophage subset markers showed increased expression of LAMP2 associated with (D) F4/80+ tissue resident and monocyte-derived RPM, (E) CD11b+ monocyte-derived RPM alone and (F) MARCO+ MZM at 2–5 days after MCAO in comparison to sham-operated animals. (G) However co-localization remained unchanged on MOMA-1+ marginal zone metallophillic macrophages (H) Co-localization of MOMA-2+ immunolabelling with LAMP-2 was high at baseline in naïve and sham-operated animals and was only significantly upregulated at 5 days after MCAO (naïve n = 4, sham n = 4, 1 day n = 4, 2 days n = 9, 5 days n = 5, 7 days n = 7). (I) Expression of protease transcripts show most proteases are modestly upregulated after MCAO; however, expression of Ctsl, which encodes Cathepsin L1, is highly upregulated 1–5 days after experimental stroke in comparison to sham-operated animals (sham n = 2, 1 day and 2 days n = 3, 5 days n = 4). (J) RT-qPCR for Ctsl confirmed upregulation of expression 2–5 days after MCAO in comparison to sham-operated animals (naïve n = 4, sham n = 4, 1 day n = 4, 2 days n = 9, 5 days n = 5, 7 days n = 7). Scale bars (C) top panel, middle panel 200 µm; (C) bottom panel 100 µm. Data show mean ± SD (B,D–H,J) *P < 0.05; ***P < 0.005; one-way analysis of variance with Bonferonni correction.