Figure 4.

Glymphatic CSF tracer influx is enhanced in mice with chronic EGG electrodes. A, Experimental timeline: mice were examined 30 d after implantation of cranial electrodes. A CSF tracer (fluorescein dextran 3 KDa) was infused via a cannula inserted into the cisterna magna of awake mice or mice under ketamine-xylazine anesthesia. Following 30 min of tracer circulation, the brain was dissected out, cut on a vibratome, and six brain sections spaced evenly across the anterior–posterior axis were analyzed for tracer influx. B, Representative images showing increased tracer penetration in mice implanted with electrodes compared with non-implanted control mice (both groups under ketamine-xylazine anesthesia). C, Quantification of tracer influx in mice under ketamine-xylazine anesthesia measured by the mean pixel intensity for each brain section showed an overall significant difference (p = 0.001) between implanted and non-implanted mice. Post hoc testing did not show significant differences between individual brain sections (error bars are SD; two-way ANOVA with Sidak's multiple comparison; n = 7). D, Average tracer influx of all brain sections was not significantly different between implanted mice and control mice (p = 0.111; Student's t test). E, Quantification of tracer influx for each brain section in awake mice showed an overall significant difference (p = 0.0001) between implanted and non-implanted mice and post hoc test showed a significant difference between the groups for most posterior brain section (error bars are SD; two-way ANOVA with Sidak's correction for multiple comparison; n = 4–6; ****p < 0.0001). F, Average tracer influx of all brain sections showed a significant difference (p = 0.0072) between implanted mice and control mice (Student's t test; n = 4–6; ****p < 0.0001). G, Representative image of tracer penetration in electrode-implanted mouse and non-implanted control mouse (both injected while awake) showing enhanced tracer penetration in the medial septum region and central cortex. H, Regional analysis of tracer penetration was performed in the lateral cortex (LC), ventral cortex (VC), and medial septum (MS). I, The greatest tracer penetration occurred in the medial septum region for all groups. Regional analysis of the medial septum region showed a significant difference between mice with electrodes and non-implanted control mice in the awake group, but no significant difference for the other regions analyzed or for mice under ketamine-xylazine anesthesia (SD, two-way ANOVA with Sidak's correction for multiple comparison; n = 4–7; ****p < 0.0001). J, Representative confocal images of CD3-expressing cells in whole-mount meninges. Scale bar, 100 μm. K, Quantification of the number of T cells in the superior sagittal sinus and in dura showed no significant difference between implanted and non-implanted animals (SD, two-way ANOVA with Sidak's multiple comparison; n = 5–6). L, Representative image of brain slice stained for AQP4. Scale bar, 200 μm. M, Quantification of AQP4 expression in the brain area depicted in L measured as mean pixel intensity did not show a difference between mice with EEG electrodes and non-implanted control mice (SD, Student's t test with Holm-Sidak correction; n = 7). N, AQP4 immunofluorescence was measured in linear regions of interest (dashed lines) across blood vessels in the same brain area and did not show significant differences between the groups (SD; multiple t tests with Holm–Sidak correction; n = 7). Scale bar, 200 μm. AU, Arbitrary units; KX, ketamine-xylazine.