Introduction

The recently discovered glymphatic and meningeal lymphatic systems are comprised of perivascular spaces and aquaporin-4 (AQP4) water channels within the brain and a network of meningeal lymphatic vessels surrounding it1. The identification of the glymphatic system and its connection to the meningeal lymphatic vessels has altered our understanding of fluid dynamics and central nervous system (CNS) waste clearance1. Like the peripheral lymphatic system of the body, the glymphatic system of the brain serves as the main pathway for the drainage of metabolic waste products, interstitial solutes, and excess fluid essential for maintaining tissue homeostasis and health. The glymphatic system uses cerebrospinal fluid (CSF) bathing the brain as a transport medium for these functions, and to distribute nutrients, metabolites, and gases. In terrestrial mammals, glymphatic clearance is optimized during sleep when the cardiac cycle, respiration, and vasomotion work together to facilitate the flow of CSF through the tissue of the brain2,3,4,5. Given its proximity and juxtaposition to the cerebral blood vasculature, glymphatic perivascular flow is mediated by the cardiac cycle and pulsation of cerebral vessels. Therefore, glymphatic fluid flow shares similar drivers to cerebral blood flow and is directly affected by changes in intracranial pressures, noradrenergic tone, ambient pressure, apnea, and hypoxia1,6. The modulations to the glymphatic system that occur during apnea and hypoxia, which are routinely experienced by diving, air-breathing tetrapods such as marine mammals, are the inspiration for this study.

The glymphatic and meningeal lymphatic pathways are comprised of distinct intersecting anatomic regions. The glymphatic system is comprised of perivascular spaces surrounding blood vessels, within the brain parenchyma, lined by astroglial AQP4 water channels1. The meningeal lymphatic system is comprised of meningeal lymphatic vessels that generally course in parallel to the major meningeal blood vasculature including dural venous sinuses that exit the skull as paired internal jugular veins (Fig.Ā 1A)5,7,8,9,10,11. The glymphatic system derives its name from the network of star-shaped astroglial cells with polarized end-feet that facilitate fluid balance and waste clearance1,12. Astroglial end-foot processes ensheathe all the blood vessels within the brain parenchyma creating an abluminal compartment between the two called the perivascular space (PVS) (also known as the Virchow-Robin space) (Fig.Ā 1B, C). The PVS within the brain is continuous with the CSF-filled subarachnoid space, thus forming a network of interconnected CSF-filled tunnels around the cerebral vascular tree that provides a low resistance path for the perfusion of CSF throughout the brain parenchyma.

Fig. 1
figure 1

Schematic representation of intracranial compartments, meningeal lymphatics, and glymphatic system components in the human. (A) Schematic sagittal section through human head displaying the intracranial compartments of brain, meningeal lymphatic vessels (MLV), and dural sinuses - superior sagittal (SSS) and transverse (TVS) - that drain to paired internal jugular veins (IJV). (B) The SSS is bounded by the periosteal (PDL) and meningeal (MDL) dural layers. Cerebrospinal fluid (CSF) influx (downward curved arrows) from the subarachnoid space (SAS), into the perivascular space (PVS) (arrowheads) occurs along penetrating arterial vessels (PAV) into brain parenchyma. Within the brain parenchyma, CSF mixes with interstitial fluid creating an admixture. This admixture effluxes into the PVS along penetrating veins and flows (upward curved arrows) into the SAS and to the periphery through MLVs and/or dural venous sinuses. AG represents arachnoid granulation and BV represents a bridging vein (BV). The dashed line represents the position of (C). (C) Morphology of the neuro-glial-vascular unit. Star-shaped astroglial cells with polarized end-feet ensheathe all the blood vessels within the brain parenchyma creating the CSF-filled PVS (arrowhead). The PVS within the brain tissue is continuous with the CSF-filled SAS.

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The influx of CSF from the subarachnoid space into the PVS occurs around cerebral arterial vessels that penetrate the brain parenchyma (Fig.Ā 1B)5,11,13,14,15,16,17,18,19,20. Within the brain parenchyma, CSF mixes with interstitial fluid creating an admixture which effluxes out of the parenchyma along cerebral veins (Fig.Ā 1B) and clears metabolic waste to the periphery through meningeal lymphatic vessels and/or dural venous sinuses (Fig.Ā 1B).

The dural venous sinuses, formed between the periosteal and meningeal layers of the dura mater, are lined by a single endothelial layer and basement membrane and lack muscular, elastic, or adventitial layers typical of other venous structures21,22,23,24. The longest dural venous sinus in humans is the superior sagittal sinus (SSS), which lies along the midline of the cranial vault (Fig.Ā 1A, B). CSF drains into the SSS for transport to the periphery through arachnoid granulations24,25,26 and by way of meningeal lymphatic vessels22,24,27,28. Arachnoid granulations (also known as Pacchionian granules and arachnoid villi) are protrusions of the subarachnoid space that perforate the inner meningeal layer of the dura and protrude into venous sinuses where they function as one-way valves for drainage of CSF into the venous circulation (Fig.Ā 1B)24.

Meningeal lymphatic vessels (MLVs) were first identified in mammals in 2015 and have since been confirmed as a CSF drainage route through advances in molecular markers and imaging capabilities including immunofluorescent labeling and magnetic resonance imaging (MRI), respectively23,25,26,29. Immunofluorescence microscopy has allowed for precise identification of MLVs within the dural layer of meninges surrounding the brain and recent studies have illuminated the importance of the MLVs in neuroimmunity and CSF circulation and clearance10,30,31. Like peripheral lymphatic vessels, MLVs express standard lymphatic identity markers such as prospero homeobox protein 1 (PROX-1), the lymphatic receptor for the extracellular matrix mucopolysaccharide hyaluronan (LYVE-1), and vascular endothelial growth factor receptor 3 (VEGFR3), which are critical to vessel development, maintenance, sprouting, and migration32,33,34. MLVs form a network of simple permeable lymphatics that drain CSF, interstitial fluid, and immune cells to larger more complex lymphatic vessels with intraluminal valves and smooth muscle that, together, propel fluid and waste towards the venous circulation for recycling. Currently, MLVs are categorized as either dorsal or basal based upon location and morphological composition30,35. Positioned near the SSS and transverse venous sinus (TVS), dorsal MLVs are characterized as simple vessels lacking valves and smooth muscle. Basal MLVs, located near the cavernous and sigmoid venous sinuses, possess relatively large lumens with intraluminal valves and are more complex. Basal MLVs have been demonstrated to be conserved primary drainage routes for CSF in both human and murine models36,37,38. Decreased drainage through MLVs has been implicated in decreased clearance of CNS waste and increased incidence of neurodegenerative disease39,40.

The glymphatic system is primarily active during sleep cycles when cardiovascular and respiratory forces work in concert to propel cerebrospinal fluid through the brain2,3,4,5. During non-REM sleep cycles, the extracellular space increases and distension of the arterial wall caused by the cardiac cycle drives CSF in a pulsatile fashion into the PVS5,11,15. Respiration imposes a negative pressure gradient, transmitted by the vertebral venous system and jugular veins, which facilitates drainage to the venous sinuses during inspiration41,42,43. Additionally, vasomotion caused by autoregulation of smooth muscle has been associated both with intraventricular CSF flow and brain clearance12.

Glymphatic dysfunction can contribute to the pathogenesis associated with traumatic and ischemic brain injuries, as well as other neurodegenerative diseases1,15,44,45,46,47,48,49. Glymphatic circulation is altered or suppressed during apnea (associated with pressures transmitted by the thoracic and vertebral veins) and by changes in vascular tone (i.e., vasodilation and vasoconstriction) and cardiac pulsatility (i.e., bradycardia and tachycardia)1,50.

To date, the glymphatic and meningeal lymphatic systems have been described in laboratory rodents, humans, and non-human primates15,51,52,53. Considering the influence of cardiorespiratory function, hypoxia, and sleep on the overall function of the glymphatic system, we chose to investigate this system in a marine mammal. Marine mammals routinely experience extended periods of breath-holding and large changes in heart rate, stroke volume, cardiac output, and peripheral tissue perfusion from diving, and can experience levels of arterial hypoxemia that would result in severe tissue damage in other mammals54,55,56,57. Moreover, certain species of marine mammals are known to regularly exhibit unihemispheric sleep57,58,59,60. Together, these traits could result in selective pressures that may influence the presence and/or structure of certain components of the glymphatic and lymphatic systems.

We investigated the glymphatic system and meningeal lymphatic vasculature in the bottlenose dolphin (Tursiops truncatus), a shallow diving cetacean. The bottlenose dolphin is arguably the best studied of all cetacean species. Many aspects of its diving physiology and functional morphology have been well characterized, as has the structure and vascular anatomy of its brain54,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78. It is also a commonly stranded species along the Atlantic coast of the United States, offering access to high-quality specimens for anatomical study79,80. Our specific goals were to determine if glymphatic and meningeal lymphatic structures, including glymphatic system-associated perivascular spaces, AQP4 water channels, and meningeal lymphatic vessels, were present in the bottlenose dolphin, and if so, to characterize their location relative to draining dural venous sinuses using a combination of immunofluorescence microscopy, histochemical staining techniques, and CT angiography. Our results demonstrate that the bottlenose dolphin possesses all the structures required for functioning glymphatic and meningeal lymphatic systems.

Results

Herein we present anatomical evidence for the presence of the structural components of the glymphatic and meningeal lymphatic system in the bottlenose dolphin (Tursiops truncatus; nā€‰=ā€‰9) including meningeal lymphatic vessels (MLVs) surrounding dural venous sinuses, and perivascular spaces (PVSs) and aquaporin (AQP4) within the parenchyma of the brain. Beginning just medial to the internal surface of the skull, we demonstrate the architecture of the intracranial dural venous sinus system based upon computed tomography (CT) and digital angiography as these structures are easily torn and therefore often lost during dissection and ex-situ examination. Dural structures were preserved in situ by immersion in formalin following bisection of the skull and brain to maintain the integrity of the dura mater and its associated sinuses allowing detailed dissection of meningeal layers and associated vasculature with minimal disruption. Then, we present the microscopic morphology of the superior sagittal (SSS) and transverse venous (TVS) sinuses including the presence of parasagittal MLVs within the dura mater using histochemical staining. Given the high degree of morphologic similarity between venous and lymphatic vessels, immunofluorescence microscopy was used to differentiate and confirm the presence of MLVs. Finally, we demonstrate the presence of astroglial AQP4 water channels and PVSs that surround penetrating cerebral blood vessels and form glymphatic pathways that transport CSF deep within the brain (see Supplemental Fig.Ā 1 for overview of methods).

The in-situ architecture of the intracranial venous sinus system surrounding the brain of the bottlenose dolphin was revealed via CT and postmortem angiography (Fig.Ā 2). The dural venous sinuses that have been well-studied in human and murine models in relation to glymphatics and meningeal lymphatics are the SSS and TVS9,10,81,82. Other intra-cranial dural sinuses include the inferior sagittal sinus, and cavernous sinus. The SSS is located deep to the cranial vault and superior to the falx cerebri that separates the cerebral hemispheres (see Supplemental Fig.Ā 1D). The SSS originates rostrally near the ethmoid bone and widens as it extends caudally towards the foramen magnum (Fig.Ā 2)83. CT angiography also revealed the characteristic curvilinear appearance of the SSS in sagittal view (Fig.Ā 2A). In transverse view the SSS appears as an inverted triangle situated at the dorsal midline (Supplemental Fig.Ā 1B-D and Fig.Ā 2B). The inferior sagittal sinus is situated deep to the SSS, along the inferior edge of the falx cerebri within the interhemispheric space above the cerebellum (Fig.Ā 2B). Each of the bilaterally-paired TVSs is located just dorsal and lateral to the foramen magnum within the posterior margin of the tentorium cerebelli (Supplemental Fig.Ā 1D). Each also displays a plexiform morphology in the dolphin with a main TVS and a more ventral linear, basket-like array of tributaries that cradles the medio-posterior cerebellum (Fig.Ā 2). The SSS, inferior sinus, and TVS intersect along the sagittal midline just above the foramen magnum to form the confluence of sinuses that ultimately coalesce and exit the skull as the vertebral venous system84. In addition, the TVS and cavernous sinus connect to the paired extracranial pterygoid sinuses and the paired internal and external jugular veins (Fig.Ā 2B). In this study, we focused our histological examination on the SSS and TVS, since these were the sinuses examined in previous human and murine studies15,51,52,53.

Fig. 2
figure 2

CT angiography digital reconstruction and schematics of the bottlenose dolphin (Tursiops truncatus) displaying the key structural features of the superior sagittal and transverse venous sinuses. (A) Left-lateral perspective CT angiography and digital reconstruction of the bottlenose dolphin (CMA 0911) reveal the overall geometry of the dural sinuses, and their intra- and extra-cranial connections. SSS represents the superior sagittal sinus, TVS represents the bilaterally paired transverse sinuses, VVS represents vertebral venous system, CS represents the cavernous sinus, EJV represents the external jugular vein, and PTY represents the bilaterally paired extracranial pterygoid venous plexus. (B) The simplified schematics in left lateral and caudal perspectives of the dural venous sinuses display positions and connections of the SSS and TVS in relation to the inferior sinus (IS) and CS, and extracranial PTY. These sinuses ultimately drain to the VVS and internal (IJV) and external (EJV) jugular veins.

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Histological staining with hematoxylin and eosin (HE) revealed that the SSS is formed by the separation of periosteal (superficial) and meningeal (deep) dural layers at the base of the falx cerebri, which partitions the left and right cerebral hemispheres (Figs.Ā 3A and 4A). The SSS possesses an endothelium and basement membrane embedded within a connective tissue stroma and is devoid of valves and a continuous smooth muscle layer (Figs.Ā 3A and 4A and B). Smooth muscle (~ā€‰4ā€“8 layers) is only present along the lateral aspect of the SSS where dorsal cerebral veins merge with and drain into the SSS (Figs.Ā 3B and 4C). The CSF-filled subarachnoid space is situated between the dural SSS and the cerebral hemispheres (Figs.Ā 3A and 4A). Arachnoid granulations are present within the dural layer positioned on the ventral aspect of the SSS, and appear as pedunculated structures comprised of a connective tissue core with a meningothelial cell lining surrounded by a capsule and subcapsular space (Fig.Ā 3C).

Fig. 3
figure 3

Light micrographs representing a transverse section of the bottlenose dolphin. (Tursiops truncatus) superior sagittal sinus displaying the histomorphology of the parasagittal structures stained with Hematoxylin and Eosin. Scale bars represent 100Ā Ī¼m. (A) Composite photomicrograph of histological section displaying the superior sagittal sinus (SSS) in transverse section (KLC 360). Boxes correspond to sites of (B), (C), and (D) in this figure. Box (E) corresponds to the site of Fig.Ā 8A. The SSS is located between the periosteal (PDL) and meningeal (MDL) dural layers, which laterally fuse around the SSS and extend ventrally to form the falx cerebri (FC) separating the cerebral hemispheres (CH). The subarachnoid space (SAS) lies between the dural SSS and CH. Arachnoid granulations (arrows) are shown perforating the dura mater. (B) Dural blood vessel (BV), in this case a dorsal cerebral vein, is shown in close juxtaposition to the lateral margin of the SSS. (C) Arachnoid granulation (AG) demonstrating the connective tissue core (CTC), meningothelial cell lining (MCL), and subcapsular space (SCS) are shown surrounded by a capsule (CAP) of arachnoid cells. (D) Located between the PDL and MDL are meningeal lymphatic vessels (arrowheads) closely clustered near a venule (V) and arteriole (A).

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Fig. 4
figure 4

Light and confocal micrographs representing a transverse section of the bottlenose dolphin (Tursiops truncatus) superior sagittal sinus displaying the histomorphology of the parasagittal structures stained with Hematoxylin and Eosin and confirmed using immunofluorescent markers PROX-1 and VEGFR3. Scale bars represent 1Ā mm (A) and 50Ā Ī¼m (B ā€“ H). (A) Composite photomicrograph of histological section demonstrating the structural arrangement of the superior sagittal sinus (SSS) in transverse section (JAX 002). Periosteal (PDL) and meningeal (MDL) dural layers, falx cerebri (FC), subarachnoid space (SAS), and cerebral hemispheres (CH) shown. Boxes correspond to the sites of (B) and (C). (B) Photomicrograph of the simple endothelial layer that lines the dorsal and ventral aspects of the SSS lumen. (C) Photomicrograph of blood vessels (BV) (arrows), in this case dorsal cerebral veins, that extend from the brain into the parasagittal dura and drain directly into SSS. Dorsal cerebral veins have a smooth muscle layer that merges with the lateral wall of SSS, which differs from the simple endothelium shown in (B). Meningeal lymphatic vessels (largest labeled MLV, others marked by arrowheads) are also present within the dura mater. Box corresponds to site of (D)-(H). (D ā€“ H) Representative confocal images (individual and merged) demonstrating and differentiating the presence and localization of blood and lymphatic vessels using immunofluorescent markers for vascular endothelial growth factor receptor 3 (VEGFR3) (green), and Prospero homeobox protein 1 (PROX-1) (magenta), with DAPI (white) counter labeling for nuclei. Note that red blood cells are auto fluorescent. Individually labeled confocal images are shown to illustrate VEGFR3 and PROX-1 localization, which can be obscured in the merged image. Arrows represent dorsal cerebral veins and arrowheads represent MLVs.

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Within the dural layers surrounding the SSS, MLVs are observed concentrated between the periosteal and meningeal layers and are usually closely associated with blood vessels (Figs.Ā 3D and 4A and C, and 5). MLVs displayed two distinct morphologies: simple and complex vessels (Fig.Ā 5). Simple MLVs appear as irregularly shaped, thin-walled vessels with a monolayer of lymphatic endothelial cells adhered to a discontinuous basement membrane and often display collapsed and narrow lumens suggestive of a low-pressure system. Occasional intraluminal valves, composed of a bilayer of lymphatic endothelium, are present within these vessels (Fig.Ā 5A). Complex MLVs display a continuous smooth muscle layer surrounding the endothelium (Fig.Ā 5A) and possess more structurally complex valves, as will be observed in the TVS below. Alternating transitions between sections of simple thin walled, and more complex meningeal lymphatic vessels were observed throughout the dural layers surrounding both the SSS and TVS.

Fig. 5
figure 5

Light and confocal micrographs representing a transverse section of the bottlenose dolphin (Tursiops truncatus) superior sagittal sinus and parasagittal dura displaying the histomorphology of the parasagittal structures stained with Hematoxylin and Eosin and confirmed using immunofluorescent markers PROX-1 and VEGFR3. Scale bars represent 100Ā Ī¼m (A) and 50Ā Ī¼m (B-P). The colored boxes in A correspond to regions shown in (B-F), (G-K), and (L-P). (A) Composite photomicrograph of histological section demonstrating the superior sagittal sinus (SSS) and parasagittal dura in transverse section (JAX 002). The dorsal periosteal layer (PDL) is composed of dense connective tissue and merges with the meningeal layer (MDL). Meningeal lymphatic vessels (MLV) are present adjacent to the SSS within the parasagittal dura. Simple MLVs (small arrowheads) appear as irregularly shaped, thin-walled vessels and often display collapsed and narrow lumens that possess intraluminal valves (small arrows). Large arrowheads point to sections of MLVs with a smooth muscle layer surrounding the epithelium. Meningeal lymphatic vessels are present and display alternating transitions between sections of simple thin-walled, and more complex, muscular walled MLVs. Complex lymphatic vessels (large arrows) display a thick continuous coat of smooth muscle cell layers surrounding the lumen, which contains a homogeneous eosinophilic substance, leukocytes, and occasional erythrocytes. (B-P) Representative confocal images (merged and individual) of the parasagittal dura in transverse section differentiating blood and lymphatic vessels using double labeling for vascular endothelial growth factor receptor 3 (VEGFR3) (green) and Prospero homeobox protein 1 (PROX-1) (magenta). DAPI (white) is used for counter labeling nuclei. (B-F) Large meningeal lymphatic vessel (MLV) (arrowhead) displaying colocalization of VEGFR3 and PROX-1 labeling and an adjacent artery (A) containing auto fluorescent red blood cells. (G-K) Simple MLVs (small arrowheads), MLVs with a smooth muscle layer surrounding the epithelium (large arrowhead), complex lymphatic vessel (arrow). (L-P) Superior sagittal sinus (SSS) and complex lymphatic vessel (arrow), are shown.

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The identity of MLVs within the parasagittal dura of the SSS was confirmed using immunofluorescence microscopy targeting lymphatic-identity proteins of PROX-1 and VEGFR3 (Figs.Ā 4D-H, and 5B-P), which were more highly conserved between humans and dolphins than LYVE-1. Positively identified MLVs displayed PROX-1 and VEGFR3 labeling of lymphatic endothelial cells lining the vessel lumen. Within lymphatic endothelial cells (LECs), Prox-1 labeling was localized to the perinuclear region, while VEGFR3 labeling was confined to the cytoplasm. Positive labeling was also observed within the smooth muscle layer of blood and lymphatic vasculature (Figs.Ā 4D-H, and 5B-P), which can be expected given their shared origins and that VEGF-C, a precursor to VEGFR3, expressed in smooth muscle cells, directs meningeal lymphangiogenesis85. PROX-1 and VEGFR3 were not identified in the endothelial lining of adjacent blood vessels (Figs.Ā 5B-F and L-P). These comparative results provide added evidence that can be used to validate the specificity of immunofluorescence labeling and reinforce the successful identification of MLVs in bottlenose dolphin meningeal tissue.

Histochemical examination of the TVS with Massonā€™s Trichrome staining highlights connective tissue and muscular elements of the blood and lymphatic vasculature embedded within the dense dural folds of the tentorium cerebelli (Fig.Ā 6). The morphology of the TVS differs from that of the SSS, where the TVS is invested with large caliber, muscular veins, separated by thin-walled venous sinuses composed of an endothelial layer and basement membrane (Fig.Ā 6A). This arrangement forms a linear ā€œbasket arrayā€ of anastomosing veins and sinus vessels that extends across the surface of the cerebellum (see Fig.Ā 2). Large caliber MLVs with intraluminal valves are present and appear to be running perpendicular to valveless muscular veins and venous sinuses (Fig.Ā 6). In the longitudinal section, MLVs surrounding the TVS display an epithelial monolayer with intraluminal valves composed of connective tissue (Fig.Ā 6B), and in some regions, smooth muscle backed by connective tissue (Fig.Ā 6C). A homogeneous fluid containing leukocytes is present within the MLV lumen (Fig.Ā 6B). The MLVs associated with the TVS are larger and appear less numerous compared to those surrounding the SSS (compare Figs.Ā 5 and 6). The identity of MLVs within the dura of the TVS was also confirmed using highly conserved immunofluorescent antibodies PROX-1 and VEGFR3 (Fig.Ā 6). Representative confocal images of merged markers display colocalized positive labeling with PROX-1 and VEGFR3 of lymphatic endothelial cells lining the lumens of MLVs near the lateral convergence of the periosteal and meningeal dural layers within the TVS (Fig.Ā 6).

Fig. 6
figure 6

Light and confocal photomicrographs of representative transverse sections of the bottlenose dolphin (Tursiops truncatus) dura mater containing the transverse sinus displaying the histomorphology of the meningeal lymphatic vessels stained with Massonā€™s Trichrome to aid in differentiating blood and lymphatic vasculature, and immunofluorescent markers to confirm the identity of meningeal lymphatic vessels, respectively. Scale bars represent 1Ā mm (A), 100Ā Ī¼m (B ā€“ C), and 50Ā Ī¼m (D ā€“ G). (A ā€“ E) KLC 360; (F) ONJ 004; (G) JAX 002. (A) Composite micrograph of the transverse sinus (TVS) displaying the position of a large caliber meningeal lymphatic vessel (MLV) within the connective tissue surrounding large muscular veins (V) and thin-walled venous sinuses (S) (KLC 360). Boxes correspond to the regions of (B) and (C). (B) Longitudinal section of an MLV displaying an epithelial monolayer with intraluminal valves (arrowheads) composed of a connective tissue base (green) present within the dural connective tissue adjacent to a large muscular vein. Within the MLV lumen there is a homogenous material (#) containing leukocytes and erythrocytes. (C) Additional section of the same lymphatic vessel depicted in (A) and (B) demonstrating an epithelial monolayer lining, and intraluminal valves (arrowheads) containing smooth muscle (purple to red) flanked by dense collagenous connective tissue (green). (D ā€“ G) Confocal images of the transverse sinus displaying merged and individual fluorescent markers, Prospero homeobox protein 1 (PROX-1) (magenta) and vascular endothelial growth factor receptor 3 (VEGFR3) (green), with and without DAPI (white) counter labeling for nuclei. (D ā€“ E) Colocalized positive labeling of lymphatic endothelial cells lining the lumen of meningeal lymphatic vessels (MLV) are shown. (F) Lymphatic vessel in cross-section with strong PROX-1 positive labeling of lymphatic endothelial cells, and intraluminal valves (arrow). VEGFR3 displays strong colocalized positive labeling of intraluminal valves. Asterisk marks adjacent MLV without intraluminal valves. (G) Asterisks mark multiple MLVs appearing as a beaded chain demonstrating PROX-1 positive labeling and co-localized VEGFR3 positive labeling (arrows) concentrated at approximately equidistant intervals along the vessel and likely represent intraluminal valves.

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The glymphatic perivascular pathway for circulating CSF into and out of the brain is juxtaposed along blood vessels that penetrate the brain tissue in the bottlenose dolphin. FigureĀ 7A displays such a vessel penetrating the surface of the cerebrum, surrounded by a distinct white ā€œhaloā€ representing the perivascular space (PVS). While the fluid normally occupying this space is typically lost during tissue processing, the anatomical compartment it would fill remains visible. Arterial and venous vasculature, found within the arachnoid and pial meninges, are continuous with the microvasculature within the parenchyma. The PVS is seen extending from the subarachnoid space along the penetrating blood vessels creating an abluminal fluid filled compartment around the cerebral vasculature (Fig.Ā 7). A distinct halo is seen surrounding the blood vessels within both the gray and white matter of both the cerebrum (Fig.Ā 7B-C) and cerebellum (Fig.Ā 7D-E), demonstrating the presence and annular morphology of the glymphatic perivascular compartment surrounding blood vessels within the brain of the bottlenose dolphin. AQP4, an astroglial water channel that facilitates glymphatic exchange, is expressed on astrocyte end-feet lining these PVSs surrounding large blood vessels throughout the brains of bottlenose dolphins (Fig.Ā 7). AQP4 is also expressed on astrocytes throughout both the gray and white matter and is enriched at astrocyte end-feet at the glia limitans and surrounding the microvasculature (Fig.Ā 8), as has been reported in the brains of both humans and rodents15,51,52,53. AQP4 is enriched around vessels and the glia limitans but it is also diffusely expressed throughout the wider brain tissue. GFAP only labels the main processes of astrocytes that contain intermediate filaments86 but AQP4 is also expressed on the wider complex and fine astrocytic processes that are not visualized by GFAP staining.

Fig. 7
figure 7

Photomicrographs of histological sections of brain parenchyma of the bottlenose dolphin (Tursiops truncatus) stained with Hematoxylin and Eosin and immunofluorescent markers for displaying the morphology of perivascular spaces and the presence of aquaporin-4 water channels lining, penetrating blood vessels. Scale bars represent 100Ā Ī¼m. (A) KLC 360; (B) TFK 020; (C) TFK 013; (D-E) KLC 360 (F) TFK 020 (G) KLC 360 (H) TFK 020 (I) TFK 013 (J) KLC 360 (K) TFK 020 (L) KLC 360. (A) Composite light micrograph demonstrating the perivascular space (PVS) (arrows) surrounding blood vessels penetrating the brain parenchyma. Larger pial blood vessels adjacent to the brain are labeled BV. (B ā€“ C) Individual light micrographs demonstrating the presence and annular morphology of PVS (arrows) within the cerebrum of two individual dolphins. (D) PVS (arrows) surrounding blood vessels penetrating the gray matter and (E) white matter of the cerebellum. (F ā€“ L) photomicrographs demonstrating immunoreactivity of the astrocyte marker glial fibrillary acidic protein (GFAP, magenta) and astroglial water channel aquaporin-4 (AQP4, green) are shown surrounding large lectin (red) positive vessels in representative standard full focus z-stack micrographs of dolphin brain tissue. (F, G) Representative images of PVSs (arrows) surrounding penetrating vessels at the brain surface are shown. (H ā€“ L) Widespread astrocytic AQP4 expression is observed throughout the brain with enrichment at astrocyte end-feet surrounding blood vessels and associated PVSs (arrows).

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Fig. 8
figure 8

Representative photomicrographs of glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), and lectin immunoreactivity in bottlenose dolphin (Tursiops truncatus) brain tissue. Scale bars represent (A) 500Ā Ī¼m and (B-F) 100Ā Ī¼m. (A) KLC 360 (B) TFK 020 (C) TFK 013 (D) TFK 020 (E) KLC 360 (F) KLC 360. (A) Representative image of a cortical gyrus and sulcus is shown with widespread AQP4 (green) expression on astrocytes (GFAP, magenta) throughout both the gray (GM) and white (WM) matter. (B) Representative sulcus and (C, D) cortical surface full focus z-stack images showing enrichment of AQP4 on astrocyte end-feet at the glia limitans (arrowheads) and surrounding the microvasculature (lectin, red) (arrows). (E, F) Higher magnification full focus z-stack images of AQP4 expression on astrocytes and perivascular AQP4 enrichment surrounding capillaries (arrows).

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Discussion

The bottlenose dolphin ā€“ a wild, large, diving mammal ā€“ possesses all the structures that are required for functional glymphatic and meningeal lymphatic systems including glymphatic and meningeal lymphatic pathways for central nervous system (CNS) waste clearance. Results reveal a structural relationship between the dural venous sinuses and meningeal lymphatic vessels (MLVs), as well as prominent glymphatic perivascular spaces (PVSs) surrounding the vasculature penetrating the brain parenchyma with widespread expression of astroglial AQP4 water channels, as has been characterized in humans and mice15,51,52,53. This morphological arrangement highlights the potential dynamic interplay between the brainā€™s blood vasculature and cerebrospinal fluid (CSF)-filled PVS, glymphatic fluid exchange, and meningeal lymphatic clearance providing a foundation for comparative studies of CSF exchange and efflux routes among marine mammals.

Marine mammal glymphatic system function is an area of study in which the intersection of morphology, physiology, and neurobiology could contribute to a broader understanding of both marine mammal diving adaptations and mammalian health. Glymphatic and meningeal lymphatic pathways are important in the context of understanding the pathophysiology of decompression sickness (marine mammals and humans) and high-altitude cerebral edema (humans), as well as other diseases that are characterized by altered CSF circulation and brain fluid dynamics (e.g., ischemic stroke) and traumatic brain injury5,11,15,39,40,46,51,87. Evidence has also pointed towards a significant role for glymphatic and lymphatic dysfunction in the pathophysiology of certain neurodegenerative diseases such as Alzheimerā€™s and Parkinsonā€™s diseases, and chronic traumatic encephalopathy (CTE) that are characterized by altered CSF dynamics46,88,89,90. The volume of CSF is dynamically linked to blood volume within the rigid and incompressible cranial cavity46,87. Therefore, any change in one volume will induce an equal and opposite compensatory change in the other. Recently, work in human subjects has shown that lowering of blood pressure through vasodilation results in an increase of intracranial pressure and reduced PVS volume, which in turn induces CSF efflux from the cranium51. Thus, it is interesting to consider hemodynamics in the bottlenose dolphin that employs a proposed pulse-transfer mechanism during breath-hold diving and locomotion to ensure equal and simultaneous arrival of arterial and venous pulses within the brain to maintain steady cerebral blood flow while diving69. Additionally, understanding the versatility of the glymphatic clearance pathway under different physiological conditions can broaden our knowledge of function beyond traditional mammalian models. For example, studies exploring how glymphatic and meningeal lymphatic system dynamics may vary during unihemispheric sleep, seen in certain species of marine mammals including bottlenose dolphins59could deepen our understanding of the systemā€™s physiological flexibility and capabilities related to clearance during sleep cycles and disturbances in humans. The effect that retrograde venous blood flow in the dolphinā€™s valveless venous system may have on glymphatic flow dynamics could also offer valuable insights about the drivers of waste clearance in marine mammals with highly variable cardiorespiratory dynamics.

The identification of glymphatic system and meningeal lymphatic morphology in bottlenose dolphins may yield insights into the adaptability of neurological pathways in marine mammals and may even shed light on mechanisms used to cope with the unique physiological challenges they face during diving. For example, studying the glymphatic system in marine mammals with differing diving capabilities (i.e., shallow vs. deep divers, short vs. long duration divers) may offer insights into understanding neuroprotective mechanisms employed during extreme breath-hold dives that can last as long as 222Ā min in beaked whales91. For instance, dive-induced centralization of venous blood could increase glymphatic flow and serve increased clearance functions during a dive. A thorough anatomic understanding of the system in varied species may also provide insights into the pathophysiology of diving-related CNS injuries in marine mammals involving blood vasculature of the brain, PVSs, dural venous sinuses, and MLVs. For example, FernĆ”ndez and colleagues (2005)87 describe severe hemorrhage within the subarachnoid space and PVSs within the brain parenchyma of beaked whales that had stranded in association with naval sonar exercises. These authors identified these lesions as part of a suite of pathologies similar to those displayed in human divers suffering decompression sickness. The distribution pattern of CNS lesions associated with decompression sickness in deep diving cetaceans88,92 mirrors glymphatic and meningeal lymphatic drainage pathways in the human brain demonstrated by MRI15,51,93 and may shed light on the pathophysiology of this disease. Such insights may also have implications for human medicine, particularly in understanding the glymphatic systemā€™s adaptability and potential relevance to neurological disorders including decompression sickness.

Meningeal lymphatic vessels (MLVs) in the bottlenose dolphin provides a direct anatomic route for drainage of CSF from the subarachnoid space within the CNS to the peripheral lymphatic system. In rodents, a nasopharyngeal lymphatic plexus has been identified as a key outflow pathway for CSF38. Given that dolphins lack a traditional olfactory system, analogous drainage likely occurs via dural lymphatics exiting the skull with cranial nerves and venous structures towards the oropharyngeal tonsils and pre-scapular lymph nodes, which are well positioned to receive cranial drainage94. Investigating these additional lymphatic routes may yield further insight into mechanisms of CNS waste clearance in diving mammals.

The role of the glymphatic and meningeal lymphatic systems in the pathophysiology of various neurologic conditions involving changes in brain fluid dynamics has been well documented. For instance, mounting evidence suggests that the glymphatic system and meningeal lymphatics play critical roles in cerebral small vessel disease95. Likewise, research in the field of Alzheimerā€™s disease may benefit from an enhanced understanding of glymphatic system function in the bottlenose dolphin ā€“ a species that has been suggested as a natural animal model for investigating beta-amyloid (AĪ²) deposition and neurofibrillary tangle formation, which are hallmarks of this disease in humans96,97. Currently, the only recognized natural animal models known to spontaneously form both pathological protein aggregates found in humans are cetaceans and pinnipeds98,99,100,101. A recent proteomic study of Alzheimerā€™s disease patients points to these molecules in plasma that are associated with indices of glymphatic and meningeal lymphatic function and are relevant in AD pathogenesis102. These markers may present a non-invasive means for comparisons with dolphins and marine mammals under human care.

Marine mammals are a polyphyletic clade of distantly related animals that are united by their shared aquatic lifestyles75,103. Three orders of mammals are included within this grouping with diverse evolutionary histories ā€“ Carnivora (which includes the marine mustelids, pinnipeds, and ursids), Sirenia (which includes dugongs and manatees), and Artiodactyla (which includes whales, dolphins, and porpoises). While cetaceans have vastly different phylogenetic histories as compared to pinnipeds, shared convergent physiological characteristics exist among these mammalian species to enhance their breath-hold diving capabilities and protect them from hypoxic insult56,62. Comparisons among cetaceans and pinnipeds might provide critical information on the evolution and adaptability of the glymphatic system to the specific physiological challenges faced by marine mammals during breath-hold diving and changes in heart rates. Cross-species comparisons could offer further insights into shared mechanisms and offer potential new therapeutic interventions for human and marine mammal medicine. For example, future studies could investigate whether glymphatic exchange in dolphins is affected by factors such as hypoxia, a common occurrence during diving, and venous retrograde flow likely occurring due to their valveless venous system. Together, the anatomical evidence of glymphatic and meningeal lymphatic systems in bottlenose dolphins opens new avenues for understanding the adaptability of this essential neurological pathway in marine mammals.

It is important to reiterate that because of the protected status of marine mammals in the US, this study relied upon the use of stranded bottlenose dolphins. Although the sex, life history category, and health status of the individuals were thus not controlled for, glymphatic and meningeal lymphatic structures were consistently observed across all individuals. Logistical challenges exist when working with protected marine mammals, including the stochastic nature of stranding events and limited access to fresh tissue. Although only fresh specimens were investigated, this study did utilize fixed tissues, and the fixation process can cause differential shrinkage of structures. For example, fixation can cause perivascular spaces to collapse to approximately one-tenth of their in vivo size in mice104. Thus, while this study demonstrates the presence of perivascular spaces within the brain of the bottlenose dolphin, the observed sizes of these spaces likely underestimate those of the living dolphin. Lastly, this study primarily focuses on anatomical aspects, and further research is needed to investigate the functional aspects of the glymphatic system in dolphins.

In summary, this work demonstrates that the bottlenose dolphin possesses the structural components necessary for functional meningeal lymphatic and glymphatic systems including MLVs in close association with dural venous sinuses surrounding the brain and the presence of PVSs and aquaporin-4 (AQP4) water channels within the brain. Results from this study provide a foundation for further investigations into the glymphatic and meningeal lymphatic system in marine mammals, particularly its functional dynamics considering the evolutionary and adaptive aspects of these critical neurological pathways. A functional understanding would provide information on the adaptability of the glymphatic system to the unique physiological challenges faced by marine mammals during breath-hold diving. As glymphatic and meningeal lymphatic dysfunction are associated with various neurological disorders in terrestrial mammals31,54,95exploring the similarities and differences in marine mammal systems may provide valuable insights for both marine mammal and human medicine. Anatomical evidence of glymphatic and meningeal lymphatic systems in bottlenose dolphins opens new avenues for understanding the adaptability of these essential neurological pathways in marine mammals and in other species routinely exposed to conditions of apnea or hypoxia.

Methods

To confirm the presence of glymphatic and meningeal lymphatic systems and identify their localization relative to the intracranial venous system (e.g., dural sinuses) our approach merged gross and microscopic techniques along with computed tomography (CT) and digital angiography. These methods were used to confirm major dural venous pathways and provide a comprehensive view of the brain glymphatic and lymphatic systems and their structural relationship to surrounding parenchyma, cerebrospinal fluid (CSF) reservoirs, and meningeal and skeletal elements. Below we highlight the individual approaches used to achieve these primary aims.

Specimens

Bottlenose dolphins (Tursiops truncatus), like all marine mammals in the US, are protected species. Thus, anatomic studies rely upon access to stranded specimens. All tissue samples used for histological and fluorescent immunolabeling techniques were obtained from stranded bottlenose dolphins (Tursiops truncatus; nā€‰=ā€‰8) collected from North Carolina beaches between 2019 and 2022; the specimen (nā€‰=ā€‰1) utilized for the CT angiography stranded in Florida in 2009 (Supplemental Table S1). Specimen collection was conducted under authorization provided by the National Marine Fisheries Service, NOAA Southeast Region Marine Mammal Stranding Agreement with UNCW. To be included in the study, carcasses must have been in fresh condition (Smithsonian Institution codes 1ā€“2)105 and have undergone a necropsy examination and histopathologic evaluation to determine sex, life history category, and health status. UNCW stranding response conducted under NOAA SE Stranding Agreement and UNCW IACUC protocols A1718-011, A2021-013, and A2324-013. Computed tomography was conducted at the University of Florida College of Veterinary Medicine under IACUC protocol 200,801,345.

Gross dissection and computed tomography of venous sinuses

Detailed dissection and CTs were employed to investigate the intracranial venous circulatory morphology and confirm major venous pathways of brain lymphatic system components. The intracranial veins were injected with a mixture of anatomic latex and contrast agents (barium sulphate or Iohexol; see84 for detailed methods), imaged, dissected at University of Florida College of Veterinary Medicine; Fig. S1C), and post-processed using Amira for FEI Systems 6.3.0 3D Data Visualization and Analysis Software for Life Sciences (https://download.amira-avizo-software.com/private/MASTERS/Amira/6.3.0/ad6fe30b/Amira-630-Windows64-VC12.exe) to examine the 3-D structure of the intracranial veins, including their associated connection and relation to skeletal landmarks. Brain lymphatic vessels are localized within the dura mater in rodents, primate, and human specimens and are most highly concentrated along venous sinuses106. Detailed dissections were further used to identify specific target areas of interest for histological comparisons among cetaceans that may not be captured by standard sampling based upon human and murine models alone.

To prepare tissues for immunofluorescence and histochemical straining, the flensed, whole head was cross-sectioned using a hand saw, at a level just rostral to the sagittal crest, and resulting sections were photo-documented (Figs. S1A, B). The rostrum was removed with the saw and the two halves of the braincase with the brain in situ were fixed in large volumes (50:1) of 10% neutral buffered formalin and stored in a refrigerated cold room at 4 ā„ƒ for a minimum of two weeks. This method allowed tissues to be fixed in situ, and subsequently sampled at specific locations, described below. Samples of the superior sagittal (SSS) and transverse venous (TVS) sinuses were systematically collected in the regions indicated by the rectangles in Fig. S1B-D. These sinuses are the routine sites of collection for humans and mice, because they are the anatomic location of some of the largest and most easily accessible meningeal lymphatic vessels in terrestrial mammals.

Histological preparation

Brain and meninges collected and fixed at necropsy as described above were sampled at multiple locations (Fig. S1B-D). Tissues were cut and oriented within cassettes and fixed inĀ 10% neutral buffered formalin for at least ten weeks, or 4% paraformaldehyde for four days and transferred to phosphate buffered saline at a pH of 7.4. Fixed samples were then processed prior to embedding in paraffin. Tissue samples were serially sectioned between 8 and 15Ā Ī¼m on either a Finesse 325 rotary microtome or American Optical 820 rotary microtome.

Histological staining and fluorescent labeling

The microscopic morphologic features of bottlenose dolphin meningeal vessels and associated lymphatic channels were investigated using standard histological staining and specialized fluorescent labeling techniques that have been established in human and murine models107,108. Standard histochemical techniques, including the use of hematoxylin and eosin (H&E), and Massonā€™s Trichrome staining, were used to identify and describe the morphology of the perivascular space and meninges and to describe their structure, and spatial relationships to surrounding tissue elements.

Confocal immunofluorescence microscopy was used to identify meningeal lymphatic vessels from paraffin embedded tissue sections. Basic Local Alignment Search Tool (BLAST, NCBI) was used to compare protein sequences between known sequences of Tursiops truncatus, Mus musculus, and Homo sapiens field-standard lymphatic endothelial cell markers. BLAST was used to identify highly conserved regions among these amino acid sequences as a preliminary metric to inform which lymphatic specific markers should be assessed via immunofluorescence (Table S2). From this analysis, we selected the antibodies utilized for the studies. Anti-mouse-PROX-1 and anti-human-VEGFR3 primary antibodies were selected for use due to the highly conserved nature of PROX-1 and VEGFR3 proteins across mammals. Samples of small intestine (Fig. S2) were collected from a subset of individuals to use as positive controls to validate that the antibodies were targeting lymphatic endothelial cells (LECs) (Fig. S3 A, B). Paraffin sections of meningeal and intestinal tissue were adhered to the slide at 60 ā„ƒ for 60Ā min and then deparaffinized using the following protocol: two 10-minute baths in Citrisolv, two 10Ā min baths in 100% ethanol, 5Ā min in 95% ethanol, 5Ā min in 75% ethanol, 5Ā min in 50% ethanol, and two 5-minute baths in phosphate buffered saline (PBS). Antigen retrieval was performed in a sodium citrate buffer in a microwave safe pressure cooker at 70% power in a 1000Ā W microwave oven. Tissue was allowed to cool to room temperature in sodium citrate antigen retrieval buffer. Slides were blocked in PBS containing 5% normal donkey serum and 0.5% Tween-20 for 4Ā h. Primary antibody diluted in blocking buffer was incubated at room temperature for 12ā€“16Ā h overnight. Samples were washed three times for 5Ā min each wash in blocking buffer to remove primary antibody. Secondary antibodies were incubated for 4ā€“6Ā h at room temperature in blocking buffer. Tissue was stained with DAPI nuclear stain for 10Ā min. Slides were mounted in ProLong Gold antifade mounting media with a 1Ā mm thick coverslip.

For AQP4, GFAP, and lectin immunofluorescence staining, paraffin sections were deparaffinized and antigen retrieval was performed in sodium citrate buffer in a steamer for 20Ā min. Slides were stained with anti-AQP4 (Millipore Sigma AB3594) and anti-GFAP (Invitrogen PA5-143587) overnight in 5% normal donkey serum in PBS and 0.3% Triton X overnight at 4 ĖšC. Slides were washed three times for 5Ā min each in PBS and 0.3% Triton X. Donkey anti-rabbit, donkey anti-goat, and Lycopersicon Esculentum (tomato) Lectin Dylight 594 (Invitrogen L32471) were incubated in 5% normal donkey serum in PBS and 0.3% Triton X for 2Ā h at room temperature. Slides were washed three times for 5Ā min each in PBS and.Ā 0.3% Triton X and mounted with Prolong Diamond Antifade Mountant with DAPI (Invitrogen P36971).

Imaging

For selected individuals, gross dissections were digitally photo-documented using a Nikon DX camera (Fig. S1B) to illustrate the presence and locations of the SSS and TVS in relation to meningeal and skeletal features, as well as the underlying brain parenchyma. All histochemical imaging was performed at the University of North Carolina Wilmingtonā€™s Richard M. Dillaman Imaging Core using a Leica Thunder Imager Tissue microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany). Immunofluorescent imaging was performed at the University of North Carolina at Chapel Hillā€™s Michael Hooker Imaging Core and Augusta University Medical College of Georgia using Olympus SM 800 and Keyence BZ-X810 confocal microscopes, respectively; images were processed using Fiji Image J2109 (https://imagej.net/downloads). Computer Aided Design software, FastCAD-32 (Evolution Computing Inc., Phoenix, AZ,Ā https://fastcad.com/main/buy.php), was used to create anatomically accurate illustrations of the brain, dural venous sinuses, and meninges to demonstrate the glymphatic and meningeal lymphatic system of T. truncatus (e.g., Fig. S1D).