Biomarkers of traumatic injury are transported from brain to blood via the glymphatic system

Abstract

The nonspecific and variable presentation of traumatic brain injury (TBI) has motivated an intense search for blood-based biomarkers that can objectively predict the severity of injury. However, it is not known how cytosolic proteins released from traumatized brain tissue reach the peripheral blood. Here we show in a murine TBI model that CSF movement through the recently characterized glymphatic pathway transports biomarkers to blood via the cervical lymphatics. Clinically relevant manipulation of glymphatic activity, including sleep deprivation and cisternotomy, suppressed or eliminated TBI-induced increases in serum S100β, GFAP, and neuron specific enolase. We conclude that routine TBI patient management may limit the clinical utility of blood-based biomarkers because their brain-to-blood transport depends on glymphatic activity.

Keywords: CSF; biomarker; clearance; lymphatic; traumatic brain injury.

Figure 1.

Glymphatic clearance of intracortical injected tracers can be suppressed with genetic, pharmacological, and mechanical manipulations. a, Time-lapse, in vivo imaging demonstrates that, subsequent to intracortical delivery, OA555 exits CNS via anterior cervical lymph vessels and accumulates in associated lymph nodes. Left, Bright-field image. Right, Epifluorescent micrographs acquired in the red emission channel (1×, 7.11× digital zoom) between 0 and 120 min following intracortical OA555 injection. Arrows indicate location of deep cervical lymph nodes. Tr, Trachea. b, Low-power confocal micrographs (4×) acquired 2 h following intracortical OA555 injection in VEGFR3-YFP mice confirm tight colocalization of OA555 signal with the highly specific lymphatic endothelial cell marker. Top, Deep cervical node with afferent lymph vessel demonstrating high VEGFR3 expression. Bottom, Efferent vessel exiting node. c–e, Epifluorescent imaging (4×) of OA555 clearance to the deep cervical lymph nodes 2 h following intracortical delivery (c) revealed no significant lateralization of fluorescence in control lymph nodes (d). When left and right nodes were pooled within individual animals, there were significantly lower node mean pixel intensities with aquaporin-4 knock-out (AQP4KO), cisterna magna cisternotomy (CMC), acetazolamide (ACZ, 20 mg/kg, i.p.) treatment, and sleep deprivation (SD) compared with control conditions (e). f, g, Liquid scintillation counting of whole-brain homogenates revealed significantly reduced 60 min clearance of ³H-dextran (f) and ¹⁴C-inulin (g) due to aquaporin-4 knock-out, cisterna magna cisternotomy, acetazolamide treatment, or sleep deprivation. All graphs represent mean ± SEM. *p < 0.05 versus control (one-way ANOVA, Tukey post hoc analysis). ***p < 0.001 versus control (one-way ANOVA, Tukey post hoc analysis). ****p < 0.0001 versus control (one-way ANOVA, Tukey post hoc analysis). ##p < 0.01 versus AQP4KO (one-way ANOVA, Tukey post hoc analysis). n.s., Not significant (paired t test). n = 4–6 mice per group.

Figure 2.

Suppression of glymphatic clearance prohibits the delivery of TBI biomarkers to the serum. a, Schematic representation of nonanesthetized, closed-head “hit and run” TBI model. b, Experimental timeline: “hit and run” TBI is induced in C57BL/6 and aquaporin-4 knock-out (AQP4KO) mice. Subgroups of C57BL/6 mice then receive cisterna magna cisternotomy (CMC), acetazolamide (ACZ, 20 mg/kg, i.p.) treatment, or sleep deprivation (SD) immediately following TBI. Serum is collected 18 h subsequent to TBI and submitted for ELISA analysis of S100β, GFAP, and NSE levels. c–e, ELISA analysis of serum levels of S100β (c), GFAP (d), and NSE (e) reveals no demonstrable differences at baseline between C57BL/6 and aquaporin-4 knock-out mice. There is a significant elevation in all three of these biomarkers of brain injury 18 h following TBI. When TBI was given in conjunction with aquaporin-4 knock-out, cisterna magna cisternotomy, acetazolamide treatment, or sleep deprivation, the concentrations of all three markers in blood were significantly reduced relative to TBI alone and were not significantly different from levels seen in injury naive mice. All graphs represent mean ± SEM. *p < 0.05 versus control (one-way ANOVA, Tukey post hoc analysis). ****p < 0.0001 versus control (one-way ANOVA, Tukey post hoc analysis). ^#p < 0.05 versus TBI (one-way ANOVA, Tukey post hoc analysis). ^##p < 0.01 versus TBI (one-way ANOVA, Tukey post hoc analysis). ^###p < 0.001 versus TBI (one-way ANOVA, Tukey post hoc analysis). ^####p < 0.0001 versus TBI (one-way ANOVA, Tukey post hoc analysis). n = 4–10 mice per group.

Figure 3.

BBB permeability is not affected by manipulations of glymphatic clearance. a, b, Fluorescent imaging of brain Evans Blue (a) reveals that there are no demonstrable differences in BBB permeability among mice receiving TBI alone and those receiving a TBI superimposed with aquaporin-4 knock-out (AQP4KO), cisterna magna cisternotomy (CMC), acetazolamide (ACZ, 20 mg/kg, i.p.) treatment, or sleep deprivation (SD) (b). All graphs represent mean ± SEM. n.s., Not significant. *p < 0.05 versus control (one-way ANOVA, Tukey post hoc analysis). **p < 0.01 versus control (one-way ANOVA, Tukey post hoc analysis). n = 4 or 5 mice per group.