Open pathways for cerebrospinal fluid outflow at the cribriform plate along the olfactory nerves

Abstract

Background: Routes along the olfactory nerves crossing the cribriform plate that extend to lymphatic vessels within the nasal cavity have been identified as a critical cerebrospinal fluid (CSF) outflow pathway. However, it is still unclear how the efflux pathways along the nerves connect to lymphatic vessels or if any functional barriers are present at this site. The aim of this study was to anatomically define the connections between the subarachnoid space and the lymphatic system at the cribriform plate in mice.

Methods: PEGylated fluorescent microbeads were infused into the CSF space in Prox1-GFP reporter mice and decalcification histology was utilized to investigate the anatomical connections between the subarachnoid space and the lymphatic vessels in the nasal submucosa. A fluorescently-labelled antibody marking vascular endothelium was injected into the cisterna magna to demonstrate the functionality of the lymphatic vessels in the olfactory region. Finally, we performed immunostaining to study the distribution of the arachnoid barrier at the cribriform plate region.

Findings: We identified that there are open and direct connections from the subarachnoid space to lymphatic vessels enwrapping the olfactory nerves as they cross the cribriform plate towards the nasal submucosa. Furthermore, lymphatic vessels adjacent to the olfactory bulbs form a continuous network that is functionally connected to lymphatics in the nasal submucosa. Immunostainings revealed a discontinuous distribution of the arachnoid barrier at the olfactory region of the mouse.

Interpretation: Our data supports a direct bulk flow mechanism through the cribriform plate allowing CSF drainage into nasal submucosal lymphatics in mice.

Funding: This study was supported by the Swiss National Science Foundation (310030_189226), Dementia Research Switzerland-Synapsis Foundation, the Heidi Seiler Stiftung and the Fondation Dr. Corinne Schuler.

Keywords: Arachnoid barrier; Cerebrospinal fluid; Cribriform plate; Lymphatic vessels; Olfactory nerves.

Fig. 1

PEG beads efficiently clear out of the CNS after lateral ventricular infusion. a and b. Representative images of 100 μm brain sections of a Prox1-GFP reporter mouse 30 min after infusion into the right lateral ventricle of 2.5 μL of unmodified beads (white) (a) and PEG beads (b). Scale bars: 1 mm. c–e. Representative pictures of PEG beads outside the CNS at 30 (c and d) and 15 min (e) after infusion. c. PEG beads (white) accumulated at the midline of the cribriform plate at the skull base. Scale bars: 500 μm. d. Afferent Prox1⁺ lymphatic vessels (green) leading to the superficial cervical lymph nodes draining PEG beads (white). Scale bars: 200 μm. e. PEG beads (white) within the deep cervical lymph nodes and efferent lymphatic vessel (green). Scale bars: 500 μm. a–e. Pictures were acquired by an AxioZoom.V16 epifluorescence microscope. Cy5 filter was used to detect the fluorescence of PEG beads (660 nm excitation/690 nm emission). Images are representative of n = 3 mice.

Fig. 2

Anatomical distribution of PEG beads within the nasal cavity. a. Decalcified coronal tissue section depicting the nasal submucosa region of a Prox1-tdTomato reporter mouse sacrificed at 45 min after i.c.v infusion. Lymphatic vessels (LVs) in red, olfactory nerve bundles (ONBs), and blood vessels are localized in the lamina propria (LP) layer of the tissue, underneath the cribriform plate (CP). Cell nuclei are shown by DAPI staining (cyan). Olfactory epithelium (OE) harbours cell bodies of Prox1⁺ olfactory sensory neurons (OSNs) in red. Scale bars: 50 μm. Acquired at the confocal microscope. b. Schematic of the sagittal plane of the mouse skull and brain showing the regions of interest within the nasal cavity. c, f, i. Representative decalcified coronal sections of the three regions of interest 1–3. Scale bars: 500 μm. d, g, j. Magnification of the nasal submucosal region showing the distribution of the PEG beads (white) with respect to the LVs in red and the nasal submucosal interstitium. Scale bars 100 μm. e, h, k. Confocal magnified views with maximum intensity projection (MIP) depicting PEG beads (white) within the lumen of the nasal submucosal lymphatics. Scale bars 10 μm. c–j. Acquired with an AxioZoom epifluorescence microscope. Images are representative of n = 5 mice. OB = olfactory bulb; CP = cribriform plate; LP = lamina propria; LVs = lymphatic vessels; ONB = olfactory nerve bundle; BVs = blood vessels; OE = olfactory epithelium. Images are representative of n = 4 mice.

Fig. 3

Visualization of PEG microbeads within lymphatic vessels crossing the cribriform plate. a. Coronal overview of a decalcified section at the cribriform plate region of a Prox1-GFP reporter mouse sacrificed at 45 min after i.c.v infusion. Lymphatic vessels (LVs) and olfactory sensory neurons (OSNs) are depicted in green, and cell nuclei (cyan) are shown with DAPI immunofluorescence staining. Scale bars 100 μm. b. Magnified view with maximum intensity projection of a lymphatic vessel crossing the cribriform plate and surrounding the olfactory nerve bundle (ONB) and blood vessel (BV). Scale bars: 50 μm. c and d. PEG beads were detected in the lumen of lymphatics (green) crossing the cribriform plate (c) and in those located in the lamina propria of the nasal submucosa (d). Scale bars: 10 μm. a–d. Acquired with the confocal microscope. Images are representative of n = 3 mice. OB = olfactory bulb; CP = cribriform plate; LP = lamina propria; LV = lymphatic vessel; ONB = olfactory nerve bundle; OE = olfactory epithelium.

Fig. 4

Lymphatic vessels crossing the cribriform plate demonstrate a loss of immunostaining for LYVE-1 towards the nasal submucosa. a. Coronal overview of a decalcified section of a Prox1-GFP reporter mouse stained with DAPI (cyan) and LYVE-1 antibody (red). Acquired by the AxioZoom epifluorescence stereomicroscope. Scale bar: 100 μm. b–e. Magnified confocal images depicting lymphatic vessel (green) crossing the CP (b), nasal septum (c), the area surrounding the nasopharynx (d), and lamina propria of nasal turbinates (e). Scale bar: 20 μm. Images are representative of n = 3 mice. ONB = olfactory nerve bundle; LV = lymphatic vessel; NP = nasopharynx; LP = lamina propria; CP = cribriform plate.

Fig. 5

APC labelled anti-CD31 antibody reveals a continuous CSF outflow pathway through lymphatic vessels at the cribriform plate to nasopharyngeal lymphatics. a. Representative sagittal view of sectioned Prox1-GFP mouse skull of the nasal cavity 50 min after injection of APC-labelled anti-CD31 antibody into the cisterna magna. Lymphatic vessels are shown in green (Prox1). Labelled antibody in magenta (CD31) was detected at the site of the cribriform plate, throughout the nasal submucosa, and in intravascular spaces in the nasopharyngeal area and the pharynx. Regions of interest b–e are marked by boxes. Scale bar: 1 mm. Sagittal regions with Prox1⁺ (green) and CD31⁺ (magenta) vessels follow from the cribriform plate via the nasal submucosa down to the nasopharynx. b. Antibody deposition in Prox1⁺ vessels and around olfactory nerves crossing the cribriform plate into the nasal submucosa (indicated by arrows, scale bar: 100 μm). c. Labelled antibody exposed a vast Prox1⁺ lymphatic vascular network stretched over the nasal submucosa (scale bar: 50 μm). d. Drainage of the labelled antibody into the nasopharyngeal area (scale bar: 50 μm) and e. further down the nasopharynx (scale bar: 100 μm). Images are representative of n = 3 mice. ONB = olfactory nerve bundle; LV = lymphatic vessel.

Fig. 6

The arachnoid is interrupted between olfactory nerve bundles and lymphatic vessels. a. Representative decalcified coronal overview of the olfactory bulbs, cribriform plate, and nasal submucosa of Prox1-tdTomato x VE-cadherin-GFP transgenic mouse immunolabeled with E-cadherin antibody. Acquired with an AxioZoom epifluorescence microscope. Scale bar: 200 μm. b–d. Magnified images of the white insert show the region between the olfactory bulbs (b), the CNS side of the cribriform plate (c), and the nasal submucosa (d). Z-stacks acquired by confocal microscope and Sum Intensity Projection were reconstructed with Fiji software. Cell nuclei are shown with DAPI staining (cyan). VE-cadherin-GFP⁺ cells (green) are detected on the surface of the olfactory bulbs (b), on the blood (BV) and lymphatic vessels (LV), and around the olfactory nerve bundles (ONBs) (c and d). E-cadherin antibody (magenta) was found on the surface of the olfactory bulbs (b), interrupted on the CNS of the cribriform plate (c), and in the nasal submucosa (d). Scale bar: 20 μm. White asterisks indicate discontinuity of the arachnoid barrier; white arrows show the arachnoid mater intact. Images are representative of n = 5 mice.

Fig. 7

Lack of a defined perineural space around olfactory nerve bundles in mice. a–d. Representative decalcified images of Prox1-tdTomato x VE-cadherin-GFP transgenic mice immunofluorescently labelled with anti-E-cadherin antibody. a and c. Pictures were taken with an AxioZoom epifluorescence microscope. Scale bars: 100 μm. b and d. Magnified confocal pictures of the white insert showing cell nuclei (blue) with DAPI staining. VE-cadherin-GFP signal (green) is visible around the olfactory nerve bundles (ONBs) and in the lymphatic vessels (LVs) (red). Epithelial cells of the arachnoid barrier and in the submucosa were detected with an E-cadherin antibody (magenta). The region on the CNS side of the cribriform plate is depicted in b; the nasal submucosa in d. Scale bars: 20 μm. Z-stacks were acquired by confocal microscope and were reconstructed with sum intensity projection (SIP) using Fiji software. White asterisks indicate discontinuity of the arachnoid barrier. Images are representative of n = 5 mice.

Fig. 8

Schematic overview of the drainage pathways alongside olfactory nerve bundles at the mouse cribriform plate region. The three meningeal layers, pia (green), arachnoid (magenta), and dura mater (dark blue), surround the OBs at the midline and at the ventral aspect of the skull. The olfactory sensory neurons (OSNs) are found at the olfactory epithelium among other cell types (not shown here). The axons of the OSNs converge into ONBs in the lamina propria and cross the cribriform plate via foramina before reaching their terminations in the OB. Blood vessels and lymphatic vessels are embedded in the lamina propria of the nasal submucosa. Here, we show a lymphatic vessel crossing at the midline of the cribriform plate, closely wrapping the ONB (see Figs. 3, 4b and 5b), and reaching the dura mater. CSF drains from the SAS along the ONBs and can enter lymphatic vessels where the arachnoid is interrupted (see Figs. 2 and 3). The pia layer ensheathing the ONB crosses the cribriform plate, and it becomes thinner once it reaches the NSM (see Fig. 7). The arachnoid is discontinuous at the cribriform plate (see Fig. 6c), and it does not seem to be present in the NSM (see Figs. 6d and 7d). Furthermore, we found that LVs crossing the plate and on the CNS side are Prox1⁺/LYVE-1⁺, while the ones in the nasal submucosa are Prox1⁺/LYVE-1⁻ (see Fig. 4), yet form a continuous network of lymphatic vessels draining the CSF towards the cervical lymph nodes (not shown here).