Meningeal Lymphangiogenesis and Enhanced Glymphatic Activity in Mice with Chronically Implanted EEG Electrodes

Abstract

Chronic electroencephalography (EEG) is a widely used tool for monitoring cortical electrical activity in experimental animals. Although chronic implants allow for high-quality, long-term recordings in preclinical studies, the electrodes are foreign objects and might therefore be expected to induce a local inflammatory response. We here analyzed the effects of chronic cranial electrode implantation on glymphatic fluid transport and in provoking structural changes in the meninges and cerebral cortex of male and female mice. Immunohistochemical analysis of brain tissue and dura revealed reactive gliosis in the cortex underlying the electrodes and extensive meningeal lymphangiogenesis in the surrounding dura. Meningeal lymphangiogenesis was also evident in mice prepared with the commonly used chronic cranial window. Glymphatic influx of a CSF tracer was significantly enhanced at 30 d postsurgery in both awake and ketamine-xylazine anesthetized mice with electrodes, supporting the concept that glymphatic influx and intracranial lymphatic drainage are interconnected. Altogether, the experimental results provide clear evidence that chronic implantation of EEG electrodes is associated with significant changes in the brain's fluid transport system. Future studies involving EEG recordings and chronic cranial windows must consider the physiological consequences of cranial implants, which include glial scarring, meningeal lymphangiogenesis, and increased glymphatic activity.SIGNIFICANCE STATEMENT This study shows that implantation of extradural electrodes provokes meningeal lymphangiogenesis, enhanced glymphatic influx of CSF, and reactive gliosis. The analysis based on CSF tracer injection in combination with immunohistochemistry showed that chronically implanted electroencephalography electrodes were surrounded by lymphatic sprouts originating from lymphatic vasculature along the dural sinuses and the middle meningeal artery. Likewise, chronic cranial windows provoked lymphatic sprouting. Tracer influx assessed in coronal slices was increased in agreement with previous reports identifying a close association between glymphatic activity and the meningeal lymphatic vasculature. Lymphangiogenesis in the meninges and altered glymphatic fluid transport after electrode implantation have not previously been described and adds new insights to the foreign body response of the CNS.

Keywords: CSF; astrogliosis; dura; glial scarring; lymphatic; meninges.

Figure 1.

Mice implanted with EEG electrodes exhibited normal sleep architecture. A, Experimental time line: 14 d after electrode implantation, mice were placed in recording chambers and allowed to habituate for 3 d followed by 3 d of continuous EEG recording. B, The weight of the mice implanted with EEG electrodes did not differ from that of non-implanted control mice (SD, Student's t test, n = 7). C, Mice implanted with electrodes and non-implanted control mice exhibited the same total duration of inactivity over the whole 24 h light/dark period (p = 0.26) and when every time point was compared (SEM, two-way ANOVA, Sidak's multiple comparison, n = 6), and (D) had equal mean durations of immobility in both the light and dark phases (SD, two-way ANOVA, Sidak's multiple comparison, n = 6). E, Mice implanted with electrodes and non-implanted control mice traveled a comparable distance over the 24 h period (SD, Student's t test, n = 6). F, Representative images of EEG trace in wakefulness, nREM sleep, and REM sleep. G, Baseline percentage time per hour of waking, nREM, and REM sleep (error bars are SEM). The data shown is derived from 48 h recordings posed on the same 24 h timeline. H, Mean percentage time spent in waking, nREM and REM sleep. Mice showed a typical light/dark difference with more wakefulness in the dark period and more nREM sleep and REM sleep in the light period (SD, two-way ANOVA, Sidak's multiple comparison, n = 5, *p < 0.05, ***p < 0.001, ****p < 0.0001). I, Average number of wake-, nREM-, and REM bouts per hour during the light and dark phases. Mice had more waking and nREM bouts during the light period (SD, two-way ANOVA, Sidak's multiple comparison, n = 5, ***p < 0.001, ****p < 0.0001). J, Mean bout duration during the light and dark periods. Mice had long bouts of wakefulness during the dark phase (SD, two-way ANOVA, Sidak's multiple comparison, n = 5, ****p < 0.0001). K, EEG power spectra of wake, nREM sleep, and REM sleep from the first 3 h of the light period (SD; n = 5). ns, non-significant.

Figure 2.

Chronic EEG electrodes induce astrogliosis and glial scarring. A, Representative image of skull and dura immunostained for the endothelial marker, CD31. Inset, Higher-magnification image of an electrode burr hole where blood vessels show the continuing presence of intact dura. B, Representative images of brain–electrode interface in coronal slices 3, 20, and 40 d after implantation. Brains were stained for GFAP (top) and CD68 (bottom). C, GFAP and CD68 immunoreactivity intensities of brain tissue at the brain–electrode interface were analyzed in rectangular ROIs (dotted line) extending inward from the brain surface. D, Mice implanted for 3, 20, and 40 d showed increased GFAP immunoreactivities under the electrodes at all tissue depths (0–700 μm) compared with control mice. Inset, GFAP staining of cortical surface 3 d after surgery (SD; two-way ANOVA with Dunnett's multiple comparison; **p < 0.01, ***p < 0.001, ****p < 0.0001). E, Three days after surgery, CD68 expression was increased at all tissue depths analyzed. At 20 and 40 d after surgery, only the tissue 0–100 μm under the pial surface showed increased CD68 expression. Inset, CD68 staining of cortical surface 3 d after surgery (SD; two-way ANOVA with Dunnett's multiple comparison; ***p < 0.001, ****p < 0.0001).

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.

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.