Anatomy and function of the vertebral column lymphatic network in mice

Abstract

Cranial lymphatic vessels (LVs) are involved in the transport of fluids, macromolecules and central nervous system (CNS) immune responses. Little information about spinal LVs is available, because these delicate structures are embedded within vertebral tissues and difficult to visualize using traditional histology. Here we show an extended vertebral column LV network using three-dimensional imaging of decalcified iDISCO⁺-clarified spine segments. Vertebral LVs connect to peripheral sensory and sympathetic ganglia and form metameric vertebral circuits connecting to lymph nodes and the thoracic duct. They drain the epidural space and the dura mater around the spinal cord and associate with leukocytes. Vertebral LVs remodel extensively after spinal cord injury and VEGF-C-induced vertebral lymphangiogenesis exacerbates the inflammatory responses, T cell infiltration and demyelination following focal spinal cord lesion. Therefore, vertebral LVs add to skull meningeal LVs as gatekeepers of CNS immunity and may be potential targets to improve the maintenance and repair of spinal tissues.

Fig. 1

Segmental pattern of the vertebral lymphatic vasculature in the thoracic spine. a Alcian blue/Alizarin red staining of the mouse vertebral column with boxes indicating position of images shown in Figs. 1–4 (thoracic vertebrae) and 5 (cervical and lumbar vertebrae), spatial orientation (A: anterior, P: posterior, L: lateral, V: ventral). b Alizarin red staining of two successive thoracic vertebrae (delimited by red/blue dots, lateral view). LF: ligamentum flavum, red asterisk: ventral vertebral body, blue arrow: facet joint (FJ), red arrowhead: ventral intervertebral disk, blue asterisk: intervertebral foramen. c latero-frontal schematic drawing corresponding to (b). DM: dura mater, LM: leptomeninges (pia mater and arachnoid), SC: spinal cord, SN: spinal nerve. d Dorsal view of LYVE1 staining. Red and blue areas correspond to two successive vertebrae. Note LVs lining ligamentum flavum. e, f Dorsal (e) and lateral (f) views of the PROX1 expression pattern. Red and blue areas correspond to two successive vertebrae. Salmon arrows: intervertebral LVs, blue arrow: dorsal LVs. g, h Segmented images of the PROX1 LV network (fronto-dorsal (g) and lateral (h) views) highlighting three successive vertebral LV units (red, blue, green). Scale bars: 2 mm (a); 300 µm (b, e, f); 200 µm (d, g, h)

Fig. 2

Modular architecture of vertebral lymphatic vasculature. a Frontal view of a cleared thoracic vertebra stained with an anti-PROX1 antibody. Red asterisk: vertebral ventral body, SC: spinal cord spatial orientation (D: dorsal, L: lateral, V: ventral). b Magnifications of red boxed areas are shown in (c–g). c Semicircular dorsal LVs (red arrow) surround the spinal cord, exit dorsolaterally (blue arrow) and also extend a latero-ventral connection to the dorsal nerve root (double red arrows in (c) and (e)). Note PROX1⁺ cells in SC and perivertebral muscles (M), FJ: facet joint. d At the ventral face of the ligamentum flavum (LF) located between two spinous processes, dorsal LVs (blue arrows) enter the vertebral canal and join semicircular LVs (red arrows). Note circles of LVs bordering the upper side of the epidural space (ES). e Ventrolateral LV circuitry around DRG (red arrowheads). Blue dotted-lines: spinal nerve roots, red dotted-lines: DRG. f Lateral view with intervertebral LVs (salmon arrows). g Two ventral branches (red arrows and arrowheads) run between each side of the ventral midline (VM) and the DRG. h Schematic representation of a frontal view of a thoracic vertebral LV unit. Longitudinal connecting vessels between vertebral units are not represented. FJ: facet joint; LF: ligamentum flavum; ES: epidural space; DRG: dorsal root ganglia; VM: ventral midline; SC: spinal cord. Black letters refer to images in (c–g). Scale bars: 300 µm (a–g)

Fig. 3

Vertebral lymphatic vessel connections with sympathetic ganglia. a, b Thoracic PROX1⁺ LVs contact paravertebral sympathetic ganglia (SG) (blue arrows). White dotted-lines: DRG. c PROX1 (white) and tyrosine hydroxylase (TH, red) double labeling shows a ventral LV branch contacting a paravertebral TH⁺ sympathetic ganglion (blue arrow). d–f PROX1 (white)/LYVE1 (red) double labeling of the LV-SG connection (blue arrow). g–i 2D-confocal images of cervical cryosections labeled with LYVE1 (white), TH (red), and DAPI (blue). White box: area magnified in (h) and (i). A LV contacts a TH⁺ SG (blue arrow). Note a second ventral LV branch running along the SG, without entering its cortical layer (h, salmon arrow). This branch is also seen in panel (b) (salmon arrow). White dotted-lines: DRG; Red asterisk: vertebral ventral body; SC: spinal cord. Scale bars: 100 µm (a–h); 200 µm (i)

Fig. 4

Variations of vLV pattern along the vertebral column. a–f Pattern of PROX1⁺ LVs in the cervical (a, b), thoracic (c, d), and lumbar (e, f) vertebral column, spatial orientation (D: dorsal, L: lateral, V: ventral). Left panels show frontal views, right panels show connection to peripheral lymph nodes (LN) and thoracic duct (TD). a, c, e Note fewer LVs in the dorsal plexus between intervertebral spinous processes of cervical and lumbar vertebrae compared to thoracic ones (blue arrows). a LVs exit bilaterally (red arrows) through the intervertebral foramen. a, c, e Also note differences in ventral root exit circuits between regions (salmon arrows). b, d, f LV ventral exit circuits (green) to b deep cervical LNs, d thoracic duct, or f to renal LNs. Red asterisk: vertebral ventral body; SC: spinal cord; Ao: Aorta. Scale bars: 300 µm (a–f)

Fig. 5

Epidural and dural lymphatic circuits of the spine. a 2D-single frontal image slice (2 µm thick) of the cervical vertebral column with enhanced brightness to reveal PROX1-expressing nuclei and spinal cord (SC), meninges including pia mater (Pi), arachnoid (A) and dura mater (DM), the epidural space (ES), and the ligamentum flavum (LF). A color-coded segmentation of layers around the spinal cord shows the meningeal layers in purple and the dura mater plus the epidural space in green. b–d 3D-reconstruction of frontal images of the cervical vertebral column with color-coded layers: the arachnoid and dura mater in purple (b); the dura mater and epidural space in green (c); combined layer marks showing the arachnoid in purple, the dura mater in white, and the epidural space in green (d), spatial orientation (D: dorsal, L: lateral, V: ventral). A noticeable LV network fills the epidural space (green) while dura mater LVs (white) are mainly restricted to DRGs (white arrows) and few branches on each side of the dorsal and ventral midline. e 3D-reconstruction of lateral images of the thoracic vertebral column with color-coded layers illustrated in (d). Blue dotted-lines: bilateral DRGs; salmon arrows: intervertebral LVs; Red asterisk: vertebral ventral body. Vx: vertebra x, Vx + 1: vertebra x + 1. f Schematic representation of the lymphatic vasculature in the thoracic vertebral column. LVs are present in the epidural space (green) around the spinal cord and in the dura mater (purple). Extravertebral LVs extend dorsal processes (blue) and ventral connections with sympathetic ganglia (SG, deep blue) and the thoracic duct (TD, light blue). Blue arrowheads; exit points of vertebral lymphatic circuits; Blue dots: connections with extravertebral lymphatic networks; Black asterisk: vertebral ventral body; DRG: dorsal root ganglia; FJ: facet joint; LF: ligamentum flavum; SC: spinal cord; SG: sympathetic ganglia. Scale bars: 300 µm (a–e)

Fig. 6

Epidural and dural lymphatic drainage. a Schematic representation of the procedure used to test spinal cord drainage: LYVE1 Ab or OVA-A⁵⁵⁵ was injected in the thoraco-lumbar (Th-Lb) region of the spinal cord parenchyme. Fifteen or forty-five minutes later, mice were sacrificed. LYVE1 was detected by labeling with a secondary antibody, while OVA-A⁵⁵⁵ was directly identified by fluorescence detection. Schema is adapted from Fig. 2c in ref. . b–e LYVE1 Ab uptake by epidural and dural lymphatic circuits after 45 min. b LYVE1 Ab (purple) injection area (black dotted line) and epidural LVs recapture (white arrows). c–e Colocalization of LYVE1 with LVs around DRG, in contact with dura mater (white arrowheads). Asterisk: vertebral ventral body, SC: spinal cord. f OVA-A⁵⁵⁵ injection leads to labeling of ipsilateral mediastinal lymph node (MeLNs) after 15 min. g Schematic representation of the lymphatic drainage pattern in the thoracic vertebral column. Thoracic vertebrae (gray), dura mater and CSF (purple), LVs of epidural space (green), extravertebral LVs and lymph node (blue). Blue dots: accumulation of OVA-A⁵⁵⁵ tracer in LVs and lymph node. MeLN: mediastinal lymph node; PvLV: paravertebral lymphatic vessel. Scale bars: 200 µm (b–e); 1 mm (f)

Fig. 7

vLVs are VEGF-C dependent and remodel after spinal injury. a–f VEGF-C induces epidural and dural lymphangiogenesis. a–c Cervical spine lymphangiogenesis after i.c.m. injection of AAV-mVEGF-C. a Schematic of i.c.m. injection to deliver AAVs into the CSF and toward the cervical spine. b, c LSFM coronal view of the cervical spine one month after AAV injection. Pattern of PROX1⁺ LVs (white) in AAV-control (b) and AAV-mVEGF-C (c) mice. Note that VEGF-C induced a robust epidural and dural lymphangiogenesis. d–f Thoraco-lumbar lymphangiogenesis induced by lumbo-sacral delivery of AAV-mVEGF-C. d Schematic of AAV injection sites into the lumbo-sacral spinal cord, adapted from Fig. 2c in ref. . e, f Pattern of PROX1⁺ LVs (white) in AAV-control (e) and AAV-mVEGF-C (f) mice. White asterisk: vertebral ventral body; SC: Spinal cord. g–i Focal injury in the thoraco-lumbar spinal cord. g Schematic of LPC injection into the thoraco-lumbar spinal cord, adapted from Fig. 2c in ref. . h, i Pattern of PROX1⁺ LVs (white) in control-non lesioned (h) and LPC-injected mice (i). Inset in (i) shows the spinal cord lesion (stippled area), spatial orientation (D: dorsal, L: lateral, V: ventral). j, k Quantification of lymphatic vessel diameter (red stippled area in (i)) after LPC-spinal cord injury in gain- and loss-of-mVEGF-C signaling mice (j) and in LPC-injured K14-VEGFR3-Ig^hom mice and -K14-VEGFR3-Ig^het (control) mice (k). n = 4 biologically independent mice/independent experiment, and data show mean+/−SD (error bar) in (j, k); one-way ANOVA with Tukey’s multiple-comparisons test (j) and Mann–Whitney U test (k); *p < 0.05, ***p < 0.001. Source data are provided as a Source Data file. Scale bars: 300 µm (b, c, e, f, h, i)

Fig. 8

Interactions of spinal LVs with immune cells. a–d Double labeling of cleared cervical vertebral column segments with antibodies against LYVE1 (green) and CD45 (purple), spatial orientation (D: dorsal, L: lateral, V: ventral). b–d Magnified images of white box in (a). Merged images (a), (d) show CD45⁺ leukocytes located along LYVE1⁺ vLVs. White asterisk: vertebral ventral body; SC: Spinal cord. e Cryosection of a cervical vertebra from a Vegfr3:YFP mouse labeled with antibodies against MHCII (red) and CD45 (white). CD45⁺ leukocytes including MHCII⁺ antigen-presenting cells are located close to and inside a YFP⁺ vLV (green) in the ligament flavum. f Quantification of CD45⁺ cells in vertebral column whole-mount preparations (see stippled area in Fig. 7i). g Cryosections of the lumbar spinal cord from LPC-injured mice previously injected with AAV-VEGFR3_4–7-Ig (LPC^control), AAV-mVEGF-C (LPC^VEGF-C) or AAV-mVEGFR-3_1–3-Ig (LPC^{VEGF-C trap}) in the lumbo-sacral region. Images representative of the ipsilateral side showing MBP⁺ myelin (green) and demyelinated area (dashed lines) with Hoechst⁺ nuclear staining (blue) in (g). h Histograms showing quantification of MBP-negative demyelinated area (dotted line in (g)) at the lesion site. Demyelinated area is increased in LPC^VEGF-C mice compared to LPC^control mice. n = 4 biologically independent mice/independent experiment, and data represent mean+/−SD (error bar); one-way ANOVA with Tukey’s multiple-comparisons test; *p < 0.05, ***p < 0.001. Source data are provided as a Source Data file. Scale bars: 300 µm (a–d); 50 µm (e); 100 µm  (g)