Figure 7. Chloroquine rapidly alkalinizes late endosomal and lysosomal compartments in meningeal phagocytes.

(A) Experimental strategy. Wild-type mice were implanted with chronic cranial windows and injected with 70 kDa dextrans systemically 2–3 weeks later. The meninges were imaged by intravital microscopy 24h later. Immediately prior to imaging, mice were injected i.p. with an acute dose of chloroquine diphosphate (500 mg/Kg).

(B) Intravital pH imaging of meningeal phagocytes before (B1) or 2 min (B2) or 8 min (B3) after chloroquine diphosphate i.p. delivery. Squares (i, ii) delineate two phagocytes before and after CQ delivery, with insets expanded in B1-i/ii, B2-i/ii, and B3-i/ii. Ratiometric images were generated for each expanded inset. Color-coded bars indicate pH range: 4.0 (red) to >6.0 (blue). Side panels in B1-i through B3-ii show individual fluorescence channels.

(C) J774A.1 murine macrophages labeled with ApHID-Alexa 546 dextrans (70 kDa), fixed and incubated in pH 5.0 buffer at 37 °C, imaged immediately following intravital imaging. A color-coded ratiometric pH image was generated as described in (B).

(D) Mean LE/Ly pH for meningeal phagocytes calculated across entire acquired stacks and averaged per mouse (n=4, 3 females and 1 male), plotted against post-CQ injection time. Bars indicate mean LE/Ly pH ± SEM. Large and small symbols correspond to values for individual mice or imaged fields within mice, respectively. Mean LE/Ly between timepoints was compared using a linear mixed model followed by Tukey's multiple comparison test (p≥0.05 n.s.; p<0.05 *; p<0.01 **).

Scale bars: 10 μm. A546: Alexa 546; CQ: chloroquine diphosphate; dex: dextran; MΦ: meningeal phagocytes; s.c.: subcutaneously; r.o.: retro-orbitally.