Figure 3.

Electrode implantation induces meningeal lymphatic sprouting. A, Overview of dura preparations. Thirty days after implantation of electrodes, mice were killed and the top of the skull with the dura in situ was removed. For higher resolution imaging of the dura alone, the dura was dissected carefully from the skull with a pair of tweezers. B, Representative image of skull and dura immunostained for the endothelial marker CD31. Inset, A burr hole and surrounding blood vessels. C, Representative image of skull and dura immunostained for the lymphatic marker podoplanin and the microglial marker Iba1. Insets, Higher magnification of increased Iba1 immunoreactivity and reorganization of podoplanin-positive vessels (arrowheads). D, Representative image of the middle meningeal artery (MMA) and the superior sagittal sinus (SSS) co-stained for prox1 GFP (green) and Lyve1 (red). Scale bar, 300 μm. Inset, Higher magnification of SSS lymphatic vessel. Scale bar, 100 μm. E, Representative image of meningeal preparation. Lymphatic vessels were mainly observed in the transverse sinus (TS) and the SSS. Scale bar, 200 μm. Inset, Higher magnification of lymphatic vessels in the TS. F, Representative images of skull and dura of control mouse with lymphatic vessels positive for the lymphatic marker Prox1 (left) and schematic image showing Prox-1-positive vessels (right). Arrowheads point to lymphatic sprouts. Lymphatic vessels were restricted to the SSS, TS, and the MMAs. G, Representative image of meningeal preparation. Lymphatic vessels underlying the bregma region paralleled and branched with the MMA. Scale bars: left, 200 μm; right, 400 μm. H, Representative images of skull and dura preparation of EEG implanted mouse with Prox1-positive lymphatic vessels showing lymphatic sprouts (arrowheads) reaching toward the electrodes. I, Quantification of the number of lymphatic sprouts showed no difference between mice with implants and non-implanted control mice (SD; Student's t test with Holm–Sidak correction; n = 5–6). J, Quantification of the lengths of lymphatic sprouts showed significantly longer sprouts in mice with EEG implants than non-implanted control mice (SD; Student's t test with Holm–Sidak correction; ***p < 0.001; n = 5–6). K, Quantification of number and length of lymphatic sprouts originating from the TS or the SSS, respectively. Significantly more sprouts initiated from the TS than the SSS in mice with implanted electrodes. Sprouts from mice with electrodes were overall longer than from control mice, but no significant difference in sprout length was found between the SSS and the TS; SD, two-way ANOVA with Sidak's multiple comparison; *p < 0.05; n = 5–6). L, Schematic representation of cranial window preparation with inset showing a representative image of the cranial window 25 d post-surgery (scalebar, 1 mm) and representative image of skull-dura preparation 25 d post-surgery. Li, Lymphatic vessels from bregma reaching the cranial window (arrowheads) and (Lii) contralateral hemisphere. M, Representative image of skull-dura preparation 35 d post-surgery. Mi and Mii, Lymphatic vessels from bregma extending to the cranial window (arrowhead) and bone regrowth.