The choroid plexus produces about a half-liter of cerebrospinal fluid daily, meaning this much must be drained from the cranial cavity into the bloodstream. A new study led by Steven Proulx, Swiss Federal Institute of Technology in Zurich, suggests that, in mice at least, this happens primarily through the lymphatic system. Using near-infrared fluorescent markers, the researchers traced the fluid’s exit routes from lymph vessels running alongside cranial nerves, to lymph nodes, and finally into peripheral blood. The study appears in the November 10 Nature Communications.
- Mouse study claims CSF clears through lymphatic vessels, not veins.
- CSF tracers reach lymph nodes quickly via vessels surrounding cranial nerves exiting the skull.
- CSF outflow slows in old mice.
"The elegant techniques are a step forward in resolving some of the problems associated with the dynamics of CSF drainage,” wrote Roxana Carare and Roy Weller, University of Southampton, U.K. (full comment below).
Scientists recognize two main paths for CSF exiting the head (Raper et al., 2016). CSF can seep out through swellings of the arachnoid mater, the middle meningeal membrane that surrounds the brain and spine, flowing into channels connected to veins. Alternatively, it can leave through lymphatic vessels apposed to nerve sheaths at their sites of exit from the skull. New reports of an active lymphatic system in the outermost meninges, the dura mater, have prompted the suggestion that these vessels may clear CSF as well (Aspelund et al., 2015; Louveau et al., 2015; Absinta et al., 2017). However, the relative contributions of these various paths and their exact routes have remained uncertain.
To get a better view of CSF drainage, first author Qiaoli Ma labeled both the CSF and the lymphatic vessels of mice. She infused a dye that fluoresces in the near-infrared coupled to a 40 kDa poly(ethylene) glycol molecule, P40D680, into the lateral ventricles of Prox1-GFP mice, which express GFP in the lymphatic vasculature (Proulx et al., 2013; Choi et al., 2011). The near-infrared dye allowed the researchers to see far into tissues with low background noise.
Surprisingly, the authors found the tracer in deep cervical lymph nodes at the earliest time they checked, 10 minutes after infusion. Looking for tracer along previously suggested exit paths, they spotted it at sites where cranial nerves, especially the olfactory and optic nerves, leave the skull. Interestingly, the recently described dural lymph vessels (Oct 17 news) lacked detectable tracer. Proulx suspects this may be because tight junctions between arachnoid mater cells isolate the CSF from the dura mater.
To follow P40D680 beyond the lymphatic system, the authors looked for near-infrared signal in veins. While lymph vessels lit up on average 11 minutes after intraventricular infusion, and lymph nodes on average 16 minutes after, the saphenous and posterior facial veins took approximately 25 minutes to light up. This indicates CSF flows first into the lymphatic system and later enters the bloodstream.
Previous studies in sheep and rabbits indicated that up to 50 percent of CSF might be cleared through lymphatic vessels (Bradbury and Cole, 1980; Boulton et al., 1998). “It’s much higher than 50 percent in our system,” Proulx told Alzforum. “We saw no early signal in blood.” Even small tracers—Evans blue, IRDye680CW, and a 3 kDa dextran coupled to AlexaFluor680—failed to transit rapidly into blood, following lymphatics-first routes similar to those of P40D680.
Wondering if the flow might change with age, the researchers compared P40D680’s travels in two- versus 18-month-old mice. Whereas the tracer reached the blood in approximately 24 minutes in the former, it took about 38 minutes in the latter.
Will the results apply to people? Weller and Carare are unsure. They noted that in mice, the arachnoid projections through which CSF is thought to clear into the bloodstream are fewer, smaller, and simpler than their counterparts in humans (Kida et al., 1993; Upton and Weller, 1985).
Proulx plans to examine CSF outflow in mouse models of Alzheimer’s disease and test stimulators of lymphatic function. A recent PET study in humans showed that CSF clearance is abnormal in AD (de Leon et al., 2017).
Carare and Weller suggested using the tracers to study how brain interstitial fluid, rather than CSF, drains along the basement membranes of cerebral capillaries and arteries, a generally accepted Aβ clearance path. They welcomed the possibility of applying the technology to humans. Proulx thinks this is feasible. “The tracers are inert … and are cleared readily through the kidney. To analyze a tracer in the bloodstream, after an injection into CSF, one need only monitor the fluorescent [near-infrared] signal of a blood vessel on the surface of the skin,” he noted. “What is missing at this point is the clinical approval to use these tracers in humans.”—Marina Chicurel
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