Fermat's Library | Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI annotated/explained version.

Although the immune-brain connection has been studied and hypothesi...

Lymphatic vessels are responsible for conducting lymph between diff...

The immune connection to neurological diseases has long been hypoth...

A lymph vessel is a thin tube that carries lymph (lymphatic flu...

The blood–brain barrier (BBB) is a highly selective semipermeable m...

Paolo Mascagni (January 25, 1755 – October 19, 1815) was an Italian...

*For correspondence: reichds@

ninds.nih.gov

†

These authors contributed

equally to this work

Competing interest:

See

page 13

Funding: See page 12

Received: 19 June 2017

Accepted: 01 September 2017

Published: 03 October 2017

Reviewing editor: Heidi

Johansen-Berg, University of

Oxford, United Kingdom

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public domain dedication.

Human and nonhuman primate meninges

harbor lymphatic vessels that can be

visualized noninvasively by MRI

Martina Absinta

1†

, Seung-Kwon Ha

1†

, Govind Nair

, Pascal Sati

Nicholas J Luciano

, Maryknoll Palisoc

, Antoine Louveau

, Kareem A Zaghloul

Stefania Pittaluga

, Jonathan Kipnis

, Daniel S Reich

Translational Neuroradiology Section, National Institute of Neurological Disorders

and Stroke, National Institutes of Health, Bethesda, United States;

Hematopathology Section, Laboratory of Pathology, National Cancer Institute,

National Institutes of Health, Bethesda, United States;

Center for Brain

Immunology and Glia, Department of Neuroscience, School of Medicine, University

of Virginia, Charlottesville, United States;

Surgical Neurology Branch, National

Institute of Neurological Disorders and Stroke, National Institutes of Health,

Bethesda, United States

Abstract Here, we report the existence of meningeal lymphatic vessels in human and nonhuman

primates (common marmoset monkeys) and the feasibility of noninvasively imaging and mapping

them in vivo with high-resolution, clinical MRI. On T2-FLAIR and T1-weighted black-blood imaging,

lymphatic vessels enhance with gadobutrol, a gadolinium-based contrast agent with high

propensity to extravasate across a permeable capillary endothelial barrier, but not with

gadofosveset, a blood-pool contrast agent. The topography of these vessels, running alongside

dural venous sinuses, recapitulates the meningeal lymphatic system of rodents. In primates,

meningeal lymphatics display a typical panel of lymphatic endothelial markers by

immunohistochemistry. This discovery holds promise for better understanding the normal

physiology of lymphatic drainage from the central nervous system and potential aberrations in

neurological diseases.

DOI: https://doi.org/10.7554/eLife.29738.001

Introduction

Recent reports (Aspelund et al., 2015; Louveau et al., 2015b) described the existence of a network

of true lymphatic vessels within the mammalian dura mater that runs alongside blood vessels, nota-

bly the superior sagittal and transverse sinuses. The dural lymphatic vessels display typical immuno-

histochemical markers that identify lymphatic vessels elsewhere in the body. They provide an

alternate conduit for drainage of immune cells and cerebrospinal fluid (CSF) from the brain, beyond

previously described pathways of flow: via arachnoid granulations into the dural venous sinuses, and

via the cribriform plate into the ethmoid region (

Weller et al., 2009). Although early reports, based

on injections of India ink into the cisterna magna of the rat, suggested that the dural pathway

accounts for only a minority of the drainage (

Kida et al., 1993), the more recent studies

(

Aspelund et al., 2015; Louveau et al., 2015b), which are based on injections of fluorescent tracers

and in vivo microscopy, indicate that the dural system may be substantially more important for drain-

age of macromolecules and immune cells than previously realized.

Whether a similar network of dural lymphatics is present in primates remains unknown. Moreover,

noninvasive visualization of the dural lymphatics – a necessary first step to understanding their

Absinta et al. eLife 2017;6:e29738. DOI: https://doi.org/10.7554/eLife.29738 1 of 15

SHORT REPORT

normal physiology and potential aberrations in neurological diseases – has not been reported. We

therefore verified pathologically the existence of a dural lymphatic network in human and nonhuman

primates (common marmoset monkeys) and evaluated two magnetic resonance imaging (MRI) tech-

niques that might enable its visualization in vivo. First, the T2-weighted fluid-attenuation inversion

recovery (T2-FLAIR) pulse sequence, which is the clinical standard for detecting lesions within the

brain parenchyma, is highly sensitive to the presence of gadolinium-based contrast agents in the

CSF (

Mamourian et al., 2000; Mathews et al., 1999; Absinta et al., 2015). Second, ‘black-blood’

imaging sequences, which are typically used for measurement of vascular wall thickness or detection

of atherosclerotic plaque, are tuned to darken the contents of blood vessels (even when they contain

a gadolinium-based contrast agent), but in the process the images may highlight vessels with other

contents and flow properties (

Mandell et al., 2017). For comparison, we also acquired a postcon-

trast T1-weighted Magnetization Prepared Rapid Acquisition of Gradient Echoes (MPRAGE) MRI

sequence, which is widely implemented for structural brain imaging and depicts avid enhancement

of dura mater and blood vessels, but which would not be expected to discriminate lymphatic

vessels.

Results and discussion

Cerebral blood vessels have a highly regulated blood-brain barrier, protecting the neuropil from

many contents of the circulating blood. Under physiological conditions, the blood-brain barrier pre-

vents gadolinium-based chelates in standard clinical use from passing into the Virchow-Robin peri-

vascular spaces and parenchyma, so that these structures do not enhance on MRI. On the other

hand, dural blood vessels lack a blood-meningeal barrier, enabling leakage of circulating fluids and

small substances, including gadolinium-based compounds. This explains the thin, though often

incomplete, dural enhancement that is seen on conventional T1-weighted MRI scans under

eLife digest How does the brain rid itself of waste products? Other organs in the body achieve

this via a system called the lymphatic system. A network of lymphatic vessels extends throughout

the body in a pattern similar to that of blood vessels. Waste products from cells, plus bacteria,

viruses and excess fluids drain out of the body’s tissues into lymphatic vessels, which transfer them

to the bloodstream. Blood vessels then carry the waste products to the kidneys, which filter them

out for excretion. Lymphatic vessels are also a highway for circulation of white blood cells, which

fight infections, and are therefore an important part of the immune system.

Unlike other organs, the brain does not contain lymphatic vessels. So how does it remove waste?

Some of the brain’s waste products enter the fluid that bathes and protects the brain – the

cerebrospinal fluid – before being disposed of via the bloodstream. However, recent studies in

rodents have also shown the presence of lymphatic vessels inside the outer membrane surrounding

the brain, the dura mater.

Absinta, Ha et al. now show that the dura mater of people and marmoset monkeys contains

lymphatic vessels too. Spotting lymphatic vessels is challenging because they resemble blood

vessels, which are much more numerous. In addition, Absinta, Ha et al. found a way to visualize the

lymphatic vessels in the dura mater using brain magnetic resonance imaging, and could confirm that

lymphatic vessels are present in autopsy tissue using special staining methods.

For magnetic resonance imaging, monkeys and human volunteers received an injection of a dye-

like substance called gadolinium, which travels via the bloodstream to the brain. In the dura mater,

gadolinium leaks out of blood vessels and collects inside lymphatic vessels, which show up as bright

white areas on brain scans. To confirm that the white areas were lymphatic vessels, the experiment

was repeated using a different dye that does not leak out of blood vessels. As expected, the signals

observed in the previous brain scans did not appear.

By visualizing the lymphatic system, this technique makes it possible to study how the brain

removes waste products and circulates white blood cells, and to examine whether this process is

impaired in aging or disease.

DOI: https://doi.org/10.7554/eLife.29738.002

Absinta et al. eLife 2017;6:e29738. DOI: https://doi.org/10.7554/eLife.29738 2 of 15

Short report Human Biology and Medicine Neuroscience

Figure 1. MRI-visualization of dural lymphatic vessels in human and nonhuman primates. In both species, conventional post-gadobutrol coronal T1-

weighted MRI is unable to discriminate lymphatic vessels due to diffuse physiological enhancement of the dura (arrows) and blood vessels, including

the superior sagittal sinus and straight sinus (arrows). On post-gadobutrol coronal T2-FLAIR and subtraction images, the dura does not enhance, and

Figure 1 continued on next page

Absinta et al. eLife 2017;6:e29738. DOI: https://doi.org/10.7554/eLife.29738 3 of 15

Short report Human Biology and Medicine Neuroscience

physiological conditions (Figure 1), as well as its abnormal diffuse or localized thickening in a variety

of pathological conditions (

Smirniotopoulos et al., 2007; Antony et al., 2015). Using high-resolu-

tion (in-plane resolution 270  270 mm or finer) T2-FLAIR and T1-weighted black-blood MRI images,

obtained after the intravenous injection of standard FDA-approved contrast material (gadobutrol),

we were able to visualize the collection of interstitial gadolinium within dural lymphatic vessels (maxi-

mum apparent diameter ~1 mm) in 5/5 human healthy volunteers and 3/3 common marmoset mon-

keys (

Figure 1). Our results suggest that in the dura, similar to many other organs throughout the

body, small intravascular molecules extravasate into the interstitium and then, under a hydrostatic

pressure gradient, collect into lymphatic capillaries through a loose lymphatic endothelium

(

Sharma et al., 2008).

To further test this hypothesis, meningeal lymphatics were also assessed using a second gadolin-

ium-based contrast agent, gadofosveset, a blood-pool contrast agent suitable for angiography

(

Lauffer et al., 1998). Gadofosveset binds reversibly to serum albumin, increasing its molecular

weight from 0.9 to 67 kDa. Under physiological conditions, albumin has a low transcapillary

exchange rate into the interstitial compartment, estimated to be on the order of 5% per hour, which

explains the propensity of gadofosveset to remain within blood vessels (

Richardson et al., 2015). In

both species, gadofosveset did not reveal dural lymphatics, especially on T1-black blood images

(

Figure 2 and Figure 2—figure supplement 1). As expected, on T1-weighted MPRAGE images,

gadofosveset provided superior intravascular enhancement, in both meningeal and parenchymal

blood vessels, compared to gadobutrol (Figure 2).

On 3D-rendering of subtraction MRI images (

Videos 1–2, Figure 1—figure supplement 1), dural

lymphatics are seen running parallel to the dural venous sinuses, especially the superior sagittal and

straight sinuses, and alongside branches of the middle meningeal artery. The topography of the

meningeal lymphatics fits with the previously described network in rodents as well as our neuropath-

ological data (

Figures 3 and 4). It is worth noting that the lymphatics visualized by MRI are large

slow-flow lymphatic ducts, whereas blind-ending and small lymphatic capillaries, clearly seen by his-

topathology (Figure 3 and Figure 3—figure supplement 1), are unlikely to be revealed by MRI. The

induction of experimental autoimmune encephalomyelitis (EAE) did not affect detection of dural lym-

phatic vessel in either of the two animals that we tested (not shown).

To support our in vivo imaging results, we further investigated the existence and topography of

lymphatics in coronal and longitudinal sections of human and marmoset dura mater. To accomplish

this, we tested a variety of putative lymphatic endothelial markers and found that selective double

immunostaining for D2-40 podoplanin/CD31 and for PROX1/CD31 was the most effective strategy

in discriminating lymphatic vs. venous blood vessels in dura samples – a challenging task since lym-

phatics sprout from transdifferentiation of venous endothelium (

Ny et al., 2005; Yaniv et al., 2006;

Srinivasan et al., 2007; Aspelund et al., 2014; Lowe et al., 2015) and persistently share some

endothelial markers. A branched network of lymphatics was clearly seen within the dura mater. On

D2-40 podoplanin/CD31 double staining, we identified a total of 93 human dural lymphatics; most

were collapsed, explaining the large range of maximum transverse diameters (range = 7–842 mm,

mean = 125 mm, standard deviation = 161 mm). The density of dural lymphatics was higher around

the venous sinuses than in more lateral areas of the dura, and higher within the meningeal layer than

the periosteal layer of the dura. As expected, red blood cells were not seen within lymphatics. In

marmosets, direct comparison between in vivo MRI and histopathology was performed

(

Absinta et al., 2014; Guy et al., 2016; Luciano et al., 2016). As shown in Figure 4 and Figure 4—

figure supplement 1, the three dural vessels detected on coronal postcontrast T2-FLAIR and on

subtraction images colocalize with three clusters of dural cells expressing the full panel of lymphatic

Figure 1 continued

lymphatic vessels (red arrows), running alongside the venous dural sinuses and within the falx cerebri, can be appreciated. Numbers refer to minutes

after the intravenous administration of gadobutrol.

DOI: https://doi.org/10.7554/eLife.29738.003

The following figure supplement is available for figure 1:

Figure supplement 1. 3D rendering of human dural lymphatics.

DOI: https://doi.org/10.7554/eLife.29738.004

Absinta et al. eLife 2017;6:e29738. DOI: https://doi.org/10.7554/eLife.29738 4 of 15

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Figure 2. Gadobutrol vs. gadofosveset in MRI-visualization of dural lymphatic vessels. Coronal T1-weighted black-blood images were acquired after

intravenous injection of two different gadolinium-based contrast agents (31 min after gadobutrol and 42 min after gadofosveset), during two MRI

sessions separated by one week. Dural lymphatics (red arrows in magnified view boxes) were better discerned using gadobutrol (standard MRI contrast

agent, which readily enters the dura) compared to gadofosveset (serum albumin-binding contrast agent, which remains largely intravascular) and were

localized around dural sinuses, middle meningeal artery, and cribriform plate (white arrows). Notably, the choroid plexus (white arrows) enhanced less

Figure 2 continued on next page

Absinta et al. eLife 2017;6:e29738. DOI: https://doi.org/10.7554/eLife.29738 5 of 15

Short report Human Biology and Medicine Neuroscience

endothelial markers (LYVE-1, D2-40 podoplanin, PROX1, COUP-TFII) and CCL21, a chemokine impli-

cated in lymphatic transmigration.

In the ongoing debate about the precise localization of lymphatics within the meninges (either

completely within the dura or ‘shared’ between the dura and the arachnoid) (

Kipnis, 2016;

Raper et al., 2016), our pathological data clearly show that at least some lymphatics are contained

entirely within the dura (

Figures 3 and 4). On limited evaluation, we were unable to visualize lym-

phatics within the leptomeninges, but additional dedicated studies, ideally with nonconventional tis-

sue-preparation methods, are warranted to fully explore this possibility. A comprehensive map of

the meningeal lymphatic network would have implications for unraveling the ways in which the men-

ingeal lymphatics participate in waste clearance and in immune cell trafficking within the central ner-

vous system (

Louveau et al., 2015a; Kipnis, 2016; Raper et al., 2016).

In inflammatory pathological conditions, cellular migration toward the dural lymphatics might be

profoundly enhanced by specific signaling and lymphatic plasticity (

Alitalo et al., 2005; Kim et al.,

2012

; Stacker et al., 2014 ). Noteworthy in this context are clusters of extravascular CD3+ lympho-

ocytes and CD68+ phagocytic meningeal macrophages that we observed in the dura of several mul-

tiple sclerosis autopsies (not shown), confirming intense immune cell trafficking and communication.

Indeed, without proper normative comparison, we cannot rule out that the extensive presence of

small lymphatics observed in multiple sclerosis dura samples is the result of inflammation-mediated

lymphoangiogenesis. On the other hand, lymphatic dysfunction might impair waste clearance in neu-

rodegenerative diseases and aging, in line with the recently captured deposition of b-amyloid in

human dura in elderly people (

Kovacs et al., 2016).

Differently from experiments implementing

injections of tracers within brain structures, here

we aimed primarily to image dural lymphatic ves-

sels in human and nonhuman primates, but we

could not prove whether dural lymphatic vessels

drain immune cells, CSF, or other substances

from the brain to deep cervical lymph nodes, nor

could we assess any link with the glymphatic sys-

tem (

Iliff et al., 2012; Xie et al., 2013;

Iliff et al., 2013). Such an analysis would proba-

bly require injection of specific MRI-detectable

tracers and acquisition of MRI time-series (not

thoroughly explored in the current work), an

important future research direction for the non-

human primate work.

Overall, our data clearly and consistently

demonstrate the existence of lymphatic vessels

within the dura mater of human and nonhuman

primates. Together with recent studies in

rodents, our results show that the meningeal

lymphatic system is evolutionarily conserved in

mammals and confirm, after exactly two centu-

ries, what the Italian anatomist Paolo Mascagni

speculated were lymphatic vessels at the surface

of human brain (

Mascagni and Bellini, 1816).

Figure 2 continued

with gadofosveset than gadobutrol, whereas meningeal and parenchymal blood vessels (both veins and arteries) did not enhance with any contrast

agent and appeared black. On conventional T1-weighted MPRAGE images, meningeal and parenchymal blood vessels enhanced with both contrast

agents, more clearly with gadofosveset.

DOI: https://doi.org/10.7554/eLife.29738.005

The following figure supplement is available for figure 2:

Figure supplement 1. MRI visualization of dural lymphatic vessels in a healthy marmoset with different contrast agents.

DOI: https://doi.org/10.7554/eLife.29738.006

Video 1. 3D-rendering of dural lymphatics (green) in a

47 year old woman from skull-stripped subtraction T1-

black-blood images (horizontal view, 180 degrees, 7

frames/minute).

DOI: https://doi.org/10.7554/eLife.29738.014

Absinta et al. eLife 2017;6:e29738. DOI: https://doi.org/10.7554/eLife.29738 6 of 15

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The ability to image the meningeal lymphatics

noninvasively immediately suggests the possibil-

ity of studying potential abnormalities in human

neurological disorders.

Materials and methods

Approvals

We carried out human studies under a protocol

(

NCT02504840) approved by NIH Institutional

Review Board. Informed consent was obtained

from all participants. Formalin-fixed human

brains were attained at autopsy after obtaining

consent from the next of kin. Animal studies

were performed under a protocol approved by

the Institutional Animal Care and Use

Committee.

Human imaging

We studied five healthy volunteers (three

women, age range 28–53 years) and obtained

scans on a 3-tesla MRI unit (Skyra, Siemens

Healthcare, Erlangen, Germany), using the body

coil for radiofrequency transmission and a 32-

element phased array coil for reception.

Prior to injection of gadolinium-based contrast agent, the following high-resolution MRI sequen-

ces were collected:

1. Whole-brain T1-Magnetization Prepared Rapid Acquisition of Gradient Echoes (MPRAGE, sag-

ittal 3D turbo-fast low angle shot [TFL] sequence, acquisition matrix 256  256, isotropic reso-

lution 1 mm, 176 slices, repetition time [TR]/echo time [TE]/inversion time [TI]=3000/3/900 ms,

flip angle 9, acquisition time 5 min 38 s);

2. Limited T2-weighted Fluid Attenuation Inversion Recovery (FLAIR, coronal 2D acquisition over

the superior sagittal sinus, field-of-view 256  256, 22 slices, reconstructed in-plane resolution

0.25 mm x 0.25 mm, 42 contiguous 3 mm slices, TR/TE/TI = 6500/93/2100 ms, echo train

length 17, bandwidth 80 Hz/pixel, acquisition time 5 min), optimized for detection of gadolin-

ium-based contrast agent in the subarachnoid space (

Absinta et al., 2015);

3. Black-blood scan (coronal 2D acquisition, Sampling Perfection with Application optimized Con-

trasts using different flip angle Evolution [SPACE] sequence, field-of-view 174  174, matrix

320  320, reconstructed in-plane resolution 0.27  0.27 mm, 64 contiguous 0.5 mm sections,

TR/TE = 938/22 ms, echo train length 35, bandwidth 434 Hz/pixel, acquisition time 7 min 50

s). A series of 2 or three overlapping coronal acquisitions were acquired to cover most of the

cerebral hemispheres;

4. Whole-brain T2-FLAIR scan (coronal 3D acquisition, SPACE sequence, field-of-view 235  235,

matrix 512  512, reconstructed in-plane resolution 0.46  0.46 mm, 176 1 mm sections, TR/

TE/TI = 4800/354/1800 ms, nonselective inversion pulse, echo-train length 298, bandwidth 780

Hz/pixel, acceleration factor 2, acquisition time 14 min);

5. Whole-brain T1-SPACE (axial 3D acquisition, acquisition matrix 256  256, isotropic resolution

0.9 mm, 112 sections, TR/TE = 600/20 ms, flip angle 120, echo-train length 28, acquisition

time 10 min).

We repeated these acquisitions after injection of gadobutrol (Gadavist, Bayer HealthCare, Whip-

pany, NJ) in all five participants. In 2 of the participants, we also repeated the entire protocol prior

to and following intravenous injection of gadofosveset (

Lauffer et al., 1998) (Ablavar, Lantheus

Medical Imaging, N Billerica, MA). Injections followed the manufacturer’s suggested dosing (0.1

mmol/kg for gadobutrol, 0.03 mmol/kg for gadofosveset).

Video 2. 3D-rendering of dural lymphatics (green) in a

6.7-year-old common marmoset from skull-stripped

subtraction T2-FLAIR images (horizontal view, 180

degrees, 7 frames/minute).

DOI: https://doi.org/10.7554/eLife.29738.015

Absinta et al. eLife 2017;6:e29738. DOI: https://doi.org/10.7554/eLife.29738 7 of 15

Short report Human Biology and Medicine Neuroscience

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Figure 3. Histopathology of human dural lymphatic vessels. (A–B) Whole-mount and coronal views of the human dura mater before sampling for

histological analysis. The dotted line in A shows where the superior sagittal sinus runs within the dural layers. (C) Coronal section of human dura stained

with H and E to highlight anatomical features of interest, including the falx cerebri and the dural venous sinus. Note the distortion of the dura after

paraffin embedding in comparison to B. (D, F, G) Within the dura mater, lymphatic and blood vessels can be differentiated using double

Figure 3 continued on next page

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Marmoset imaging

We studied three healthy adult common marmosets (Callithrix jacchus) (one female, two males, age

range 4–11 years). After the baseline MRI, experimental autoimmune encephalomyelitis (EAE) was

induced in 2 marmosets with 0.2 mg of fresh-frozen human white matter homogenate as previously

described (

Gaita

n et al., 2014). Marmosets were placed in a sphinx position within the magnet, and

scans were obtained on a 7-tesla MRI unit (Avance AVIII, Bruker BioSpin, Billerica, MA, USA). Data

acquisition was performed in transmit-only/receive-only mode using a homemade linear birdcage

coil (120 mm inner diameter) as a radiofrequency transmitter and a homemade, 8-channel radio-fre-

quency surface-array receiver coil assembly placed over the head of the animal. Prior to injection of

contrast agent, we collected:

1. Whole-brain T1-weighted MPRAGE scan (coronal 3D acquisition, Modified Driven Equilibrium

Fourier Transform [MDEFT] sequence, in-plane voxel size 0.15 mm x 0.15 mm, 36 contiguous 1

mm sections, TR/TE/TI = 12.5/4/1200 ms, flip angle 12 degrees, 2 segments of 1800 ms, acqui-

sition time 7 min);

2. Whole-brain T2-FLAIR scan (coronal 2D acquisition, Rapid Acquisition with Relaxation

Enhancement [RARE] sequence, voxel size 0.15 mm x 0.15 mm, 36 contiguous 1 mm sections,

TR/TE/TI = 10,000/36/2500 ms, flip angle 90–180 degrees, 2 averages, acquisition time 13

min).

We performed the same scans following intravenous injection of gadobutrol and gadofosveset, in

two different MRI sessions. Injections used single (0.1 mmol/kg for gadobutrol, 0.03 mmol/kg for

gadofosveset) or triple the recommended human dosing (0.3 mmol/kg for gadobutrol, 0.09 mmol/

kg for gadofosveset).

Image processing

Scanner-generated DICOM images were converted into NIFTI files for postprocessing. Using MIPAV

software (

https://mipav.cit.nih.gov), precontrast scans were rigidly registered to postcontrast scans,

and subtraction images were created for anatomical identification of dural lymphatic vessels. 3D

skull-stripped subtraction images were then imported into Osirix software for maximum intensity

projection (MIP) 3D rendering. The same postprocessing was performed for scans acquired after

gadofosveset injection, and a direct comparison between the two gadolinium-based contrast agents

was made.

Neuropathological evaluation of human and primate dural lymphatic

vessels

Neuropathological evaluation focused on human and marmoset brain dura mater samples. Multiple

human dura samples were obtained from 2 formalin-fixed brains (60- and 77-year-olds with long-

standing progressive multiple sclerosis) and from a 33-year-old with refractory epilepsy undergoing

anterior temporal lobectomy (

Figure 3—source data 1). Primate samples were obtained from 3

adult common marmosets (2 with EAE) [Figure 4—source data 1]. After general anesthesia and

transcardial perfusion of 4% paraformaldehyde, marmoset brains were extracted and stored in 10%

Figure 3 continued

immunostaining for PROX1 (a transcription factor involved in lymphangiogenesis, nuclear staining) and CD31 (a vascular endothelial cell marker). (E, H–

K) Similarly, lymphatic and blood vessels can be differentiated using immunohistochemical (E, H, I) and immunofluorescent (J, K) double staining for

podoplanin (D2-40, endothelial membrane staining) and CD31. Red blood cells are seen within blood vessels, but not within lymphatic vessels. Insets

(F–I) were rotated relative to the original figures in D and E. Abbreviations: H and E: hematoxylin and eosin; LV: lymphatic vessels; BV: blood vessels.

DOI: https://doi.org/10.7554/eLife.29738.007

The following source data and figure supplements are available for figure 3:

Source data 1. Table of human tissue sampling.

DOI: https://doi.org/10.7554/eLife.29738.010

Figure supplement 1. Histopathology of human dural lymphatic vessels.

DOI: https://doi.org/10.7554/eLife.29738.008

Figure supplement 2. Positive and negative control staining for lymphatic vessels.

DOI: https://doi.org/10.7554/eLife.29738.009

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Figure 4. Histopathology of dural lymphatic vessels in common marmosets. MRI-histopathological correlation in a 4.4-year-old marmoset (A–F). (A, B)

Postcontrast coronal T2-FLAIR showing three enhancing lymphatic vessels within the dura, and corresponding H and E section for anatomical reference

(area of interest in the box). (C-F) Three clusters of cells (circles), surrounding the SSS and positive for lymphatic endothelial cell markers, correspond to

the three enhancing areas seen on MRI (A). LYVE-1 is a lymphatic endothelial cell marker (membrane staining), PROX1 and COUP-TFII are transcription

factors involved in lymphangiogenesis (nuclear staining), and CCL21 is a chemokine implicated in lymphatic transmigration. Higher magnifications are

shown in

Figure 4—figure supplement 1. 10.3-year-old marmoset (G-I). (G) H and E coronal section showing the brain parenchyma, meninges, and

area of interest for lymphatics for anatomical reference (box). This animal did not recover after undergoing general anesthesia for blood tuberculosis

Figure 4 continued on next page

Absinta et al. eLife 2017;6:e29738. DOI: https://doi.org/10.7554/eLife.29738 10 of 15

Short report Human Biology and Medicine Neuroscience

formalin. For primate brains, MRI-matched histological sections were achieved via 7T MRI of the

fixed brain and subsequent gross sectioning with an individualized, MRI-designed, 3D-printed cut-

ting box, as previously described (

Absinta et al., 2014; Guy et al., 2016; Luciano et al., 2016).

Coronal and longitudinal 3 to 7 mm paraffin sections of the dura mater were stained with hema-

toxylin and eosin (H and E). Immunohistochemical and immunofluorescent analysis for classical lym-

phatic endothelial cell markers [lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1),

podoplanin (D2-40), prospero homeobox protein 1 (PROX1), COUP transcription factor 2 (COUP-

TFII) and CCL21], were performed on representative slides. Selective double staining for lymphatic

vs. vascular endothelial markers (D2-40/CD31 and PROX1/CD31) were also executed on representa-

tive sections and implemented for histological quantification; the maximum diameter was recorded

for each lymphatic vessel, and descriptive statistics were computed. We also stained for lymphocytes

(CD3) and monocytes/macrophages (CD68). Five-mm paraffin sections of the human skin were used

as positive controls for lymphatic endothelium cell marker assessment (

Figure 3—figure supple-

ment 2

). For comparison, 10 paraffin human brain tissue blocks, selected for the presence of nearly

intact leptomeningeal architecture, were implemented to assess the presence of lymphatics within

the subarachnoid space and cerebral parenchyma.

Antibodies

Source, antibody type, and dilution are indicated sequentially as follows: LIVE-1 (Abcam, UK, rabbit

polyclonal, 1:200); podoplanin D2-40 (AbD Serotec, Hercules, CA, mouse monoclonal, 1:50); PROX-

1 (AngioBio, San Diego, CA, rabbit polyclonal, 1:50); COUP TFII (R&D Systems,

Minneapolis, MN, mouse monoclonal, 1:200); CCL21 (Abcam, rabbit polyclonal, 1:200); CD31

(Abcam, rabbit polyclonal, 1:50); CD68 KP1 (Thermofisher, Waltham, MA, mouse monoclonal,

1:100); CD3 (Dako, Santa Clara, CA, rabbit polyclonal, 1:200); goat anti-mouse Alexafluor 488 IgG

(Invitrogen, Carlsbad, CA, 1:250) and goat anti-rabbit Alexafluor 594 IgG antibodies (Invitrogen,

1:250).

Immunohistochemistry

Immunostaining was performed on 3 to 7 mm-thick consecutive paraffin sections with antibodies to

LYVE1, D2-40 podoplanin, PROX1, COUP-TFII, CCL21, CD31, CD68, CD3 and visualized with 3,3’-

diaminobenzidine (DAB). Briefly, after deparaffinization, sections were rinsed in triphosphate-buff-

ered saline (TBS) for 10 min each, processed for antigen retrieval, and treated in 3% hydrogen per-

oxide for 10 min. Sections were blocked with a protein-blocking solution for 20 min and incubated

with the primary antibodies overnight at 4

C or 1 hr at room temperature, then rinsed and incubated

with secondary antibodies for 30 min. The immunoreaction was visualized with DAB. After washing,

sections were counterstained with 10% hematoxylin. Stained sections were visualized and digitized

(Axio Observer Z.1, Carl Zeiss Microscopy, NY, USA). Selective double staining for lymphatic and

vascular endothelium markers (D2-40 podoplanin/CD31, PROX1/CD31), as well double staining

for D2-40 podoplanin/PROX1,were also executed on representative sections. Double staining was

performed using, in sequence, DAB horseradish peroxidase (HRP) and Vector Blue alkaline

Figure 4 continued

testing, and at necropsy a stroke was identified in one hemisphere (asterisk). (H-I) Lymphatic (circles) and blood vessels were differentiated using

double staining for podoplanin (D2-40, endothelial membrane staining) and vascular endothelial cell marker (CD31), respectively. 3.7-year-old marmoset

(J-L). (J) Whole-mount of the marmoset dura, including the SSS. The high level of vascularization of the dura can be appreciated. The arrow indicates

the area shown in K and L. (K, L) Double staining for podoplanin D2-40 for lymphatics, and CD31 for vascular endothelial cells, show the presence of a

linear vascular structure parallel to the SSS, positive for podoplanin D2-40 but not CD31. The SSS is positive for both markers, probably because of

antibody entrapment during immunofluorescence staining of the whole-mount dura. Abbreviations: H and E: hematoxylin and eosin; LV: lymphatic

vessels; SSS: superior sagittal sinus.

DOI: https://doi.org/10.7554/eLife.29738.011

The following source data and figure supplement are available for figure 4:

Source data 1. Table of marmoset tissue sampling.

DOI: https://doi.org/10.7554/eLife.29738.013

Figure supplement 1. Histopathology of marmoset dural lymphatic vessels.

DOI: https://doi.org/10.7554/eLife.29738.012

Absinta et al. eLife 2017;6:e29738. DOI: https://doi.org/10.7554/eLife.29738 11 of 15

Short report Human Biology and Medicine Neuroscience

phosphatase (AP) methods. As negative control, sections of human skin, human dura mater, and

marmoset brain were stained without the primary antibodies; no background staining and/or non-

specific binding was noted (

Figure 3—figure supplement 2).

Immunofluorescence

After deparaffinization, sections were treated using the same method as described above. Sections

were incubated with a cocktail of antibodies to D2-40 podoplanin and CD31 for 2 hr at room tem-

perature, then rinsed and incubated with secondary antibodies with Alexafluor 488 goat anti-mouse

(Invitrogen, dilution 1:250) and Alexafluor 594 goat anti rabbit IgG antibodies (Invitrogen, dilution

1:250), respectively, for 30 min at room temperature. After washing, sections were mounted with

DAPI Immuno Mount. Stained sections were visualized and digitized (Axio Observer Z.1, Carl Zeiss

Microscopy, NY, USA).

Acknowledgements

The Intramural Research Program of NINDS supported this study. We thank the study participants,

Irene Cortese and the NINDS Neuroimmunology Clinic for patient recruitment and care, the NIH

Functional Magnetic Resonance Imaging Facility for MRI support, Afonso C Silva and his laboratory

for access to the 7T animal scanner and customized hardware, Steven Jacobson and Emily Leibovitch

for collaboration on our animal studies, and Roger Depaz for assistance in animal preparation.

Additional information

Competing interests

Martina Absinta: Dr. Absinta was partially supported by a National Multiple Sclerosis Society (NMSS)

fellowship award #FG 2093-A-1 and holds a Marilyn Hilton Award for Innovation in MS research from

the Conrad N. Hilton Foundation. Daniel S Reich: Dr. Reich received research support from collabo-

rations with the Myelin Repair Foundation and Vertex Pharmaceuticals, unrelated to the present

study. The other authors declare that no competing interests exist.

Funding

Funder Grant reference number Author

National Institute of Neurolo-

gical Disorders and Stroke

Intramural Research

Program

Martina Absinta

Seung-Kwon Ha

Govind Nair

Pascal Sati

Nicholas J Luciano

Kareem A Zaghloul

Daniel S Reich

National Multiple Sclerosis So-

ciety

Postdoctoral Fellowship Martina Absinta

Conrad N. Hilton Foundation Marilyn Hilton Award for

Innovation in Multiple

Sclerosis Research

Martina Absinta

National Cancer Institute Intramural Research

Program

Maryknoll Palisoc

Stefania Pittaluga

National Institute on Aging R01AG034113 Antoine Louveau

Jonathan Kipnis

Lymphatic Education and Re-

search Network

Postdoctoral fellowship Antoine Louveau

The funders had no role in study design, data collection and interpretation, or the

decision to submit the work for publication.

Absinta et al. eLife 2017;6:e29738. DOI: https://doi.org/10.7554/eLife.29738 12 of 15

Short report Human Biology and Medicine Neuroscience

Author contributions

Martina Absinta, Conceptualization, Data curation, Formal analysis, Methodology, Writing—original

draft, Writing—review and editing; Seung-Kwon Ha, Govind Nair, Pascal Sati, Antoine Louveau, Ste-

fania Pittaluga, Data curation, Formal analysis, Writing—review and editing; Nicholas J Luciano, Kar-

eem A Zaghloul, Data curation, Writing—review and editing; Maryknoll Palisoc, Formal analysis,

Methodology; Jonathan Kipnis, Conceptualization, Writing—review and editing; Daniel S Reich,

Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation,

Methodology, Writing—original draft, Project administration, Writing—review and editing

Author ORCIDs

Martina Absinta, https://orcid.org/0000-0003-0276-383X

Seung-Kwon Ha, http://orcid.org/0000-0003-3671-4454

Govind Nair, http://orcid.org/0000-0003-3725-615X

Kareem A Zaghloul, https://orcid.org/0000-0001-8575-3578

Daniel S Reich, https://orcid.org/0000-0002-2628-4334

Ethics

Human subjects: We carried out human studies under a protocol (NCT02504840) approved by the

NIH Institutional Review Board. Informed consent was obtained from all participants.

Animal experimentation: Animal studies were performed under a protocol approved by the Institu-

tional Animal Care and Use Committee of the NIH (NIH Animal Studies Protocol 1308-15).

Decision letter and Author response

Decision letter https://doi.org/10.7554/eLife.29738.017

Author response https://doi.org/10.7554/eLife.29738.018

Additional files

Supplementary files

Transparent reporting form

DOI: https://doi.org/10.7554/eLife.29738.016

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Discussion

The immune connection to neurological diseases has long been hypothesized. Many of the significant biomarkers (SNPs, genes) for immune function and autoimmune diseases are also connected to neurologic disease. Many researchers believe that figuring out how to target and manipulate these cells could help treat various neurological disorders like Alzheimer’s disease, migraines or injuries to the brain. A lymph vessel is a thin tube that carries lymph (lymphatic fluid) and white blood cells through the lymphatic system. Lymph travels around the body through a network of vessels in much the same way that blood travels around the body through the blood vessels. Whereas the blood carries nutrients and other substances into our tissues, the lymph vessels drain fluid from the tissues and transport it to the lymph nodes. These small gland-like masses of tissue filter out and destroy bacteria and other harmful substances. The bigger lymph vessels then carry the cleaned fluid back to the vein called the superior vena cava, where it enters the blood stream. To learn more about lymphatic vessels, read on here: https://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0022680/ ![Imgur](https://i.imgur.com/lw3CaGV.jpg) The blood–brain barrier (BBB) is a highly selective semipermeable membrane barrier that separates the circulating blood from the brain and extracellular fluid in the central nervous system (CNS). https://en.wikipedia.org/wiki/Blood%E2%80%93brain_barrier ![Imgur](https://i.imgur.com/hxHApNl.png) Paolo Mascagni (January 25, 1755 – October 19, 1815) was an Italian physician, known for his study of human anatomy, in particular for the first complete description of the lymphatic system. It's pretty incredible that he saw lymphatic vessels in the brain 200 years before it's experimental confirmation. ![Imgur](https://i.imgur.com/Ckv5rsE.png) Lymphatic vessels are responsible for conducting lymph between different parts of the body. Lymphatic drainage is important for maintaining fluid homeostasis and providing a means for immune cells to traffic into draining lymph nodes from other parts of the body, allowing for immune surveillance of bodily tissues. Before this discovery, it was thought that the mammalian CNS did not contain a lymphatic system and relied upon alternative routes such as the glymphatic system, a cerebrospinal fluid (CSF) drainage pathway under the cribriform plate and into the lymphatics of the nasal mucosa, and arachnoid granulations to clear itself of excess protein, fluid, and metabolic waste products. Although the immune-brain connection has been studied and hypothesized for over a hundred years, this is the first imaging evidence that our brains may drain waste out through lymphatic vessels, the body’s sewer system. These results suggest the vessels could act as a pipeline between the brain and the immune system. The presumed absence of CNS lymphatics (before this discovery) was an important pillar in the long-held dogma that the CNS is an immune-privileged tissue to which immune cells have incredibly restricted access under normal physiological conditions (due to the brain-blood barrier).

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