A new perspective on cerebrospinal fluid dynamics after subarachnoid hemorrhage: From normal physiology to pathophysiological changes

Abstract

Knowledge about the dynamic metabolism and function of cerebrospinal fluid (CSF) physiology has rapidly progressed in recent decades. It has traditionally been suggested that CSF is produced by the choroid plexus and drains to the arachnoid villi. However, recent findings have revealed that the brain parenchyma produces a large portion of CSF and drains through the perivascular glymphatic system and meningeal lymphatic vessels into the blood. The primary function of CSF is not limited to maintaining physiological CNS homeostasis but also participates in clearing waste products resulting from neurodegenerative diseases and acute brain injury. Aneurysmal subarachnoid hemorrhage (SAH), a disastrous subtype of acute brain injury, is associated with high mortality and morbidity. Post-SAH complications contribute to the poor outcomes associated with SAH. Recently, abnormal CSF flow was suggested to play an essential role in the post-SAH pathophysiological changes, such as increased intracerebral pressure, brain edema formation, hydrocephalus, and delayed blood clearance. An in-depth understanding of CSF dynamics in post-SAH events would shed light on potential development of SAH treatment options. This review summarizes and updates the latest physiological characteristics of CSF dynamics and discusses potential pathophysiological changes and therapeutic targets after SAH.

Keywords

Subarachnoid hemorrhage cerebrospinal fluid meningeal lymphatic drainage glymphatic system blood clot

Introduction

Cerebrospinal fluid (CSF) is a clear, colorless fluid that fills the space around/within the brain and spine. It was first revealed in the 19th century that the CSF was secreted by the choroid plexus within the ventricles, collected in the arachnoid villi, and absorbed by the superior sagittal sinus.¹ Since then, the CSF was believed to primarily function as a physical buffer and waste sink for the central nervous system (CNS).^1,2 Ongoing advancements have broadened our understanding of the physiological role of CSF in brain metabolism and function. The CSF is reportedly produced by the choroid plexus and the brain parenchyma.^2,3 Arachnoid villi may not be the only pathway of CSF drainage due to the newly identified meningeal lymphatic drainage⁴ and glymphatic system.^5,6 CSF circulation participates in maintaining CNS homeostasis, which stabilizes intracerebral pressure (ICP), cerebral blood perfusion, solute (e.g. glucose, ions, and proteins) concentration, and pH levels.^7,8 Conversely, CSF is responsible for transporting nutrients and hormones, as well as waste protein (e.g. beta-amyloid and tau) clearance via its dynamic fluid circulation.⁹ Given the critical physiological function of CSF, emerging evidence has shown that CSF circulation was impaired in both chronic neurodegenerative disorders and acute brain injury, which may contribute to the pathology of disease progression and poor outcome.^3,5,9–11

Subarachnoid hemorrhage (SAH), often caused by ruptured cerebral aneurysm, is a life-threatening subtype of stroke that results from bleeding into the subarachnoid space. The mortality rate of SAH patients ranges from 8.3%–66.7%.¹² SAH survivors often have a decreased quality of life due to the high incidence (nearly 45%) of neurological and cognitive impairment.¹³ Neurological deficits following SAH primarily result from the initial injury and secondary pathological response.^9,14 The initial injury, also known as the first part of early brain injury (EBI), occurs in the first 72 h after SAH and is characterized by increased ICP, as well as reduced cerebral perfusion pressure (CPP) and cerebral blood flow (CBF), inevitably leading to global ischemia.¹⁵ This ischemia-induced hypoxic state further results in mitochondrial failure in neurons and glial cells, and initiates cytotoxic edema formation and cell death.¹⁶ In addition, ischemia also leads to astrocytic and microglial activation and endothelial cell death, further triggering an inflammatory response and blood-brain barrier (BBB) dysfunction.¹⁷ Ionic homeostasis disruption, excitotoxicity, and oxidative stress occur simultaneously after initial ischemia.^17–19 In this process, toxic products of subarachnoid blood (e.g. hemoglobin, heme, Fe²⁺) and EBI metabolites (e.g. Damage-associated molecular patterns (DAMPs), cytokines) are transported by CSF movement, and further aggravate the environment within and around the brain. This contributes to the secondary injury after SAH, which is considered the second part of EBI. The secondary injury reportedly lasts for 2–4 weeks after SAH, and leads to delayed neuronal death, neuroinflammation, cerebral vasospasm, and chronic hydrocephalus.²⁰

Undoubtedly, ischemia and toxic products of subarachnoid blood are responsible for the poor outcomes following SAH. Abnormal CSF flow is suggested to be an important participant of both short- and long-term pathophysiological sequelae of SAH, such as increased ICP, decreased CPP, brain edema formation, as well as acute and chronic hydrocephalus.¹⁷ Meanwhile, the clearance of blood and its lysis products by the CSF circulation is considered an important mechanism of self-repair in the brain.²¹ While surgical interventions, such as lumbar drainage and external ventricular drainage, are widely used to accelerate SAH blood clearance in the clinical setting, the mortality and morbidity remain high.²² An in-depth understanding of the role that CSF dynamics have in SAH pathological process would shed light for SAH treatment. In this review, we summarize and update the physiological characteristics of CSF dynamics, and discuss potential pathophysiological changes and therapeutic targets after SAH.

Physiology of CSF dynamics in normal brain

CSF formation

Currently, there are two known sources of CSF production within the brain: the choroid plexus system and the brain parenchymal system.^2,9,11,23 The choroid plexus system, considered the main source of CSF production, is reportedly responsible for secreting nearly 80% of CSF.^2,9,24 Recently, the bulk fluid exchange, which occurs between the brain interstitial fluid (ISF) and the capillaries within the brain parenchyma, has also been proposed to participate in CSF formation.²⁵ In the following section, we will briefly introduce the roles of these two systems in CSF formation (Figure 1).

Figure 1.

CSF production. a) The BBB and brain parenchyma are currently considered critical components of CSF formation. A large amount of fluid is exchanged bi-directionally among blood, CSF, and interstitial fluid within the perivascular space and the glymphatic system contributes to the CSF production. b) Traditionally, CSF is primarily produced by the choroid plexus in cerebral lateral ventricles, comprising cuboidal epithelium and fenestrated capillary endothelium. A variety of transporters on the basolateral (plasma side) and apical (CSF side) membranes of choroid plexus epithelium facilitate CSF formation. The apical membrane is occupied by the Na+-K+-ATPase, K+ channels (Kv1.3; Kv1.1 and Kir7.1), Cl- conductances (VRAC and Clir), potassium cotransporters NKCC1 (Na+-K+-2Cl−) and KCC4 (K+-Cl−), Na+/H+ exchanger (NHE1), Na+-HCO3− cotransporters NBCE, and water channel aquaporin (AQP)1. Transporters on the basolateral membrane of the choroid plexus epithelium are the K⁺-Cl⁻ cotransporter, KCC3, Na⁺-HCO3⁻ cotransporters (NBCn and NCBE), Cl⁻/HCO3⁻ exchanger AE2, glucose transporter-1 (GLUT1), and AQP1.

Choroid plexus system

Choroid plexuses are located in the cerebral ventricular system, which remain the relatively well-known anatomical structure responsible for CSF production.²³ The choroid plexuses are an epithelial-endothelial unit consisting of central capillaries with fenestrated endothelium and cuboidal epithelium.²⁴ The cuboidal epithelial cells on ventricular site stay tightly connected with the assistance of the tight junction (TJ) proteins, which controls the diffusion of excess fluid and plasma contents. The main TJ proteins include the occludins, claudins, and Zonula occludens (ZO) proteins.²⁴

Due to different hydrostatic pressure and solutes (ions, pH, and proteins) found between CSF and plasma,⁹ hydrostatic pressure and osmotic gradients between the blood, choroid plexus epithelial cells, and CSF are the proposed potential drivers of CSF secretion.³ However, CSF formation is found relative insensitive to the alteration of ICP or plasma osmolarity.² This insensitivity is considered may be caused by the existence of many ion and water transporters found on the choroid plexus epithelial cells.²⁴ These transporters are divided into basolateral (plasma side) and apical (CSF side) transporters according to their facing side.^23,24 The apical membrane is occupied by Na⁺-K⁺-ATPase, K⁺ channels (Kv1.3; Kv1.1, and Kir7.1), Cl^– conductances (VRAC and Clir), potassium cotransporters (NKCC1 (Na⁺-K⁺-2Cl⁻) and KCC4 (K⁺-Cl⁻)), Na+/H+ exchanger (NHE1), Na⁺-HCO³⁻ cotransporters NBCE, and water channel aquaporin AQP1. Conversely, fewer transporters are found on the basolateral membrane of the choroid plexus epithelium, including the K⁺-Cl⁻ cotransporter (KCC3), Na⁺-HCO³⁻ cotransporters (NBCn and NCBE), Cl⁻/HCO³⁻ exchanger (AE2), glucose transporter-1 (GLUT1), and AQP1.²⁴

As these transporters are easily affected by the pathological changes that occur after acute brain injury, we will briefly introduce mechanisms of transporter-mediated CSF formation in choroid plexuses. Accordingly, the Na⁺, Cl^–, HCO3^–, and Cl^– movements initiate CSF formation. The uptake of Na⁺ on the choroid plexus epithelium of the basolateral side is driven by the Na⁺ gradient, and contributes to the primary step of CSF formation.^3,26 Na⁺-HCO³⁻ cotransporters, such as NBCn and NCBE, facilitate intracellular accumulation of Na⁺ and HCO3^–, which accompany H⁺ and Cl^– outflow.^26,27 Carbonic anhydrases within the choroid plexus epithelium provide intracellular HCO^3– and H⁺ from H₂O and CO_2, which has been proposed to be directly associated with HCO^3– transporters (e.g. AE2 and NBCE) on two sides to maintain CSF pH.²⁸ Cl^– uptake from plasma is also dependent on the basolateral transporter, AE2,²⁹ which is further transported by Cl^– conductances and Cl^–-related cotransporters into CSF.³⁰ The apical Na⁺-K⁺-ATPase drives the Na⁺ efflux and K⁺ influx in the choroid plexus epithelium, which provides the gradient for Na⁺ influx from plasma and secondary K⁺ transport.³¹ Meanwhile, the luminal NKCC1 and NHE also participate in the choroid plexus-contributed Na⁺ in CSF.³² The high cellular K⁺ is transported outward, which is driven by the transmembrane chemical K⁺ gradient through the KCC3 and KCC4.³³ The luminal K⁺ channels also facilitate the recycling of CSF K⁺ from the choroid plexus.³⁴

Using the osmotically driven force between plasma and CSF formed by abundant ion transporters, AQP1 facilitates transcellular water transfer.³⁵ Besides, the potential water transport on the basolateral membrane may be accompanied by ion movement with transporters, such as NKCCs and KCCs. The AQP7 also reportedly participates in transcellular water transfer.²³ Additionally, it should be noted that the water and cations are reportedly transported by a paracellular pathway due to the stranding connection of TJs.³⁰ This form of paracellular claudin connection leads to pore formation, which allows water and cations to cross.³⁶

Brain parenchyma system

The brain parenchymal system has been proposed as a source of CSF production, as the CSF produced from the trans-endothelium fluid exchange and glymphatic CSF-ISF fluid exchange in the brain parenchyma.² Traditionally, the BBB was considered an endothelial cell-based barrier responsible for mediating the exchange of ions, nutrients, and cells between the blood and the brain parenchyma.³⁷ However, the detailed mechanisms involved in substance exchange at the cellular and molecular levels remain ambiguously described. It was believed that the capillary endothelium has high electrical resistance and low permeability to polar solutes due to its tight connection.³⁸ Upon the discovery of ion channels and transporters on the brain side, its function was further extended to include substance transportation, such as ions, water, larger molecules, and even cells located between blood and CSF.³⁷

Recently, the BBB was revealed to be more than an independent unit of endothelial cells. As the “neurovascular unit”, the BBB includes pericytes, astrocytes, smooth muscle cells, microglia, and neurons.^16,39,40 The capillary-astrocyte complex of the neurovascular unit has been proposed as the site of CSF and brain ISF production and exchange. ISF fills the extracellular space between neurons and glia in the brain parenchyma. The CSF-ISF fluid exchanges in the perivascular space are driven by the osmotic forces (i.e. ion and protein gradients) between the blood, perivascular space, and brain parenchyma.^25,41 While the water has been proposed to be filtered from the high hydrostatic arterial vessels to the ISF and CSF, this phenomenon is reversed when the ISF and CSF reaches the low hydrostatic pressure of capillaries and venules.⁶ This blood-CSF-ISF fluid exchange system is known as the “cerebral glymphatic system”.^6,10 We will discuss its detailed mechanism in the following sections.

CSF absorption

Both CSF formation and absorption play important roles in regulating CSF volume. Similar to the divergent presumptions on CSF formation, the current view on CSF absorption also remains under debate. According to the anatomical location, features, and functional capabilities, several hypotheses were proposed, including arachnoid villi drainage, meningeal lymphatic drainage, and glymphatic drainage.^2,3,9,42,43 (Figure 2(a))

Figure 2.

CSF absorption and glymphatic system. a. CSF absorption. The majority of CSF directly drains into the arachnoid villi along the superior sagittal sinus into the blood. Some CSF drains along the paraneural sheaths of olfactory nerves across the cribriform plate and nasal submucosa to the nasal lymph nodes. The meningeal lymphatic vessels distributed on the dorsal and basal dura (green vessels) also facilitate CSF drainage into cervical lymph nodes. b. Glymphatic system. The subarachnoid CSF communicates with parenchymal ISF in the perivascular space around the penetrating pial vessels. The hydrostatic gradient between the arterial and venous perivascular space drives continuous bulk CSF-ISF exchange from the arterial side to the venous side. Polarized AQP4 water channels located on the astrocytic endfeet around arteries and veins facilitate CSF movement from periarterial spaces into the brain parenchyma, and from the brain parenchyma into perivenous spaces.

Arachnoid villi drainage

Arachnoid villi (i.e. arachnoid granulations) are small protrusions that extend from the arachnoid mater into the dural venous sinuses located on the outer membrane of the dura mater. The discovery of arachnoid villi drainage resulted from an experiment conducted by Quincke, who injected cinnabar into the CSF of animals and found accumulation around arachnoid granulations.⁴⁴ Colored tracers injected into CSF consistently pass the arachnoid villi to the sagittal sinus, and eventually travel to internal jugular veins.⁴⁵ Arachnoid villi are considered as an anatomical structure with “one-way valve”, depending on the hydrostatic pressure gradient between CSF and dural venous blood.^3,44 Particles up to 7.5 μm in size were found to pass through the arachnoid villi and enter blood in monkeys.⁴⁶ The potential transmembrane transport may rely on endothelial pinocytosis and vacuolization, as well as extracellular cistern.⁴⁶ It is necessary to ensure that there is a 3–5 mmHg hydrostatic pressure gradient between CSF and dural venous blood to maintain CSF drainage from arachnoid villi.^8,43 However, the exact changes related to hydrostatic pressure dynamics under physiological and pathological conditions remain unclear due to the difficulties of replicating the dynamic changes of ICP in an in vitro model.⁴⁷

Lymphatic drainage

Numerous studies have corroborated that CSF injection of tracer could be found not only in cervical lymphatics,^41,48–50 but also in spinal extradural lymphatic vessels⁵¹ in mammals. The reported percentage of CSF-lymphatic drainage ranges from 5–50%^48–50 across several preclinical studies. The discrepancy may result from differences in molecular weights of the tracers, access time after injection, and experimental methods. The routes that mediate lymphatic drainage include the perineural sheaths of cranial and spinal nerves,⁵² as well as the meningeal lymphatic vessels.⁵³

Experimental tracers have been shown to move along the olfactory nerve (considered the major nerve drainage pathway⁵⁴) with CSF, and appear sequentially in the cribriform plate,⁵⁵ nasal submucosa, and the nasal lymph nodes.⁵⁶ This movement was present during the inpiration phase, but stopped in expiration phase of respiratory.⁵⁷ The potential explanation may be that the altered ICP and venous pressure was associated with respiration. However, current research regarding the driving force between perineural subarachnoid space and lymphatics remains limited, but further exploration of anatomy and physiology in the cribriform plexus and the perineural pathway may promote the understanding of this area.

Recently, numerous studies have revealed that lymphatic vessels exist within the dura meninges in rodents, humans, and non-human primates.^4,58–62 Fluid, immune cells, macromolecules, and antigens in the CSF could be transported via lymphatic vessels of the meninges toward the deep cervical lymph nodes.^4,61,62 These intracranial lymphatic networks express all of the molecular hallmarks of lymphatic endothelial cells, such as homeobox protein 1 (PROX1), vascular endothelial growth factor receptor 3 (VEGFR3), lymphatic endothelial hyaluronan receptor (LYVE)-1, podoplanin, and C-C motif chemokine ligand (CCL) 21.^4,58 Similar to peripheral lymphatics, there is a high correlation between meningeal lymphangiogenesis and the expression of VEGFR3 and vascular endothelial growth factor C (VEGFC), the growth factors secreted by blood vessel smooth muscle cells.^42,63 Impairment of meningeal lymphatic drainage is shown to impede the diffusion of CSF tracer into the perivascular spaces and the brain parenchyma, as well as the clearance of intraparenchymal tracer.⁶⁰

Interestingly, the meningeal lymphatic vessels display distinct junctional organization on the basal and dorsal sides.⁴² The dorsal meningeal lymphatic vessels shared similar characterization with peripheral lymphatic vessels, and are proposed to be responsible for the transport of macromolecules and immune cell in brain.^4,58,64,65 In contrast, the basal meningeal lymphatic vessels move on the skull base, and are proposed to maintain unidirectional lymph flow toward the deep cervical lymph nodes.^53,58,63 Due to its proximity to the basal subarachnoid space, the basal lymphatic network is believed to contribute more in CSF clearance from brain parenchyma and ventricular system.^53,58 Additionally, some studies have suggested that the CSF collected from olfactory nerves is also transported by the basal lymphatic network to the deep cervical lymph nodes.^4,42,52 However, it remains unclear the molecular mechanisms underlying CSF-meningeal lymphatic exchange and the connective anatomy of the meningeal-extracranial lymphatic network.

Glymphatic drainage

Fluid exchange exists among blood, CSF, and ISF. According to the newly-defined glymphatic system, CSF communicates with ISF in the brain parenchyma through the perivascular space (will be discussed in the following section).⁶⁶ Water and solutes are proposed to move across the endothelium in a bidirectional manner driven by the osmotic and hydrostatic forces. Because of the unavailable methodology in monitoring such fluid exchange, as well as the difficulty in quantification, it remains unclear if, or how much, CSF is drained by the glymphatic system. Overall, this theory is akin to a theoretical hypothesis of CSF drainage,⁹ which warrants further exploration.

CSF movement and glymphatic system

Traditionally, CSF movement is believed to originate from the choroid plexus and epithelium in the lateral ventricles, which then travels through the third and fourth ventricle, passes into the subarachnoid space, and is then absorbed in the arachnoid villi.^7,9 This unidirectional pattern of CSF flow is now being challenged by the glymphatic CSF-ISF fluid exchange system, in which CSF flow is bidirectional.^10,11 The glymphatic system is named due to its functional similarity to the peripheral lymphatic system and the importance of astrocytic AQP4 in this pathway.^66,67 Using either two-photon imaging or light sheet fluorescence microscopy for visualizing the small fluorescent tracer movement in subarachnoid space, it was revealed that the bulk of the CSF in the subarachnoid space enters the brain parenchyma along perivascular spaces around pial-perforating arteries, and flows out of the brain parenchyma through the perivenous space (the perivascular space also named Virchow-Robin space).^67,68 The AQP4 channels located on the perivascular astrocytic endfeet facilitated this flux in fluid exchange¹⁷ (Figure 2(b)). The CSF movement in the glymphatic system is suggested to be driven by the hydrostatic gradient between the arterial and venous perivascular spaces, and is affected by arterial pulsatility and respiration, as well state of consciousness and head position.^69–73

Of interest, the perivascular fluid flux highly correlates with the molecular weight and size of the substances. Intracisternally infused small single-domain antibodies (17 kDa; 4.5 nm diameter of hydration) accessed perivascular basement membranes more readily than large full-length immunoglobulin G antibodies (150 kDa; 10 nm diameter of hydration).⁷⁴ The lower molecular weight and diameter of hydration tracers allowed them to enter the brain parenchyma much more easily, as demonstrated by the contrast-enhanced MRI.⁷⁵ This may potentially be due to the tight connection between astrocytic endfeet around the vessel basement membranes, in which only 20-nm clefts between overlapping processes can provide direct communication with the interstitium.⁷⁶

Accumulating studies report that the glymphatic system participates in the maintenance of metabolism⁷⁷ and clearance of waste products, such as beta-amyloid⁶⁴ and ɑ-Synuclein⁷⁸ in brain parenchyma. However, the research pertaining to the glymphatic system is still in its infancy. The questions that remain unanswered include: 1) whether ISF drainage can directly cross the venules, the dural lymphatics, or arachnoid villi; 2) which methods can distinguish the perivascular space around the venules and arterioles, as well as the fluid exchange form; 3) whether fluid exchange stays identical between substances with different molecular weights; 4) and what are the other fluid exchange molecular mechanisms besides AQP4 participation.

CSF dynamics after SAH

SAH is characteristically described as blood that influxes into the subarachnoid space, mixes with the CSF throughout the entire circulation system of brain CSF, and diffuses throughout the brain. In humans, the first three days after SAH are the most critical and life-threatening.⁷⁹ Cognitive deficits develop in the clinical setting 6–14 days after SAH.⁸⁰ Current knowledge regarding pathological changes after SAH is mainly derived from pre-clinical studies. The brain injury is divided into four phases: acute phase (0–24 hours post-hemorrhage), sub-acute phase (1–3d post-hemorrhage), delayed phase (3–14d post-hemorrhage), and chronic phase (>14d post-hemorrhage).²⁰ In the acute phase, early vasospasm, acute hydrocephalus, increased ICP, and decreased CBF account for the initial damage.¹⁵ When SAH progresses to the sub-acute phase, the blood and its degradation products within the subarachnoid space are detrimental, not only in the tissue proximal to the vasculature, but also in the parenchyma.²⁰ In the delayed and chronic phases, the blood clot in the subarachnoid space begins to be visibly cleared. While brain water content, CBP, and ICP return to baseline,⁸¹ delayed vasospasm, neuronal death, secondary inflammatory response, and hydrocephalus appear in this period.²⁰ Besides, both acute and chronic pathological changes after SAH are associated with the CSF dynamic disorder that involves CSF production, movement, and drainage. A plethora of studies report pathological impairment found in the choroid plexus,⁸² BBB,⁸³ arachnoid granulations,⁸⁴ lymphatic,⁸⁵ and glymphatic system,¹⁷ which may be related to post-SAH blood clearance. (Figure 3)

Figure 3.

Pathological CSF circulation after SAH. a. Blood, with its lysis products, fulfill the subarachnoid space and ventricular system with CSF circulation after SAH. b. Toxic products of blood stimulates choroid plexus epithelium facilitating AQP1 expression and CSF hypersecretion. c. Fibrin/fibrinogen fill in the arachnoid villi and block the arachnoid villi CSF drainage. Erythrocytes were found to drain with CSF to the cervical lymph nodes. d. Blood clot with fibrin occupying the perivascular space inhibits CSF/ISF exchange. Dysfunctional endothelium results in plasma ions and protein extravasation, which promotes water influx to the perivascular space. Dislocated astrocytic AQP4 further leads to parenchymal water accumulation.

CSF production responses to SAH

Choroid plexus system responses to SAH

SAH is associated with a 30-50% incidence rate of visualized intraventricular hemorrhage (IVH).^86,87 Blood, with its products of degradation that are toxic to the choroid plexus and epithelial cells, are inevitably carried by the CSF flowing from the bleeding site to the ventricular system in both the acute and chronic phases.⁸⁸

Several clinical studies have reported ependymal damage and loss in the fetal or neonatal post-hemorrhagic periventricular areas.^89,90 Preclinical research further demonstrated the acute choroid plexus cell death and ultrastructural changes that occur within 3 days after IVH in preterm rabbits and hemoglobin/heme-treated in vitro model.⁹¹ Moreover, acute choroid plexus and ependymal death were also consistently found in the adult rat model of IVH ^92,93 and the rabbit model of SAH.⁹⁴ The inflammatory responses and caspases activation, as well as intracellular inclusions (IgG), accompanied cell death after hemorrhagic insults in neonatal and adult animal models.^91–94 In addition, the choroid plexus atrophy and ependymal loss commonly exists in the chronic phase (1 week to 3 months) after IVH and SAH in various animal models, such as pigs⁹⁵ and rabbits.^96,97 The post-hemorrhage fibrin deposition, which induces thrombosis and inflammatory changes in the vessel wall,⁹⁵ as well as choroidal artery vasospasm⁹⁶ are considered the primary contributors of these pathological changes.

IVH is a risk factor in the development of hydrocephalus and poor prognosis after SAH⁹⁸ Hydrocephalus developed in 52-62% of SAH patients with IVH.^98,99 Using the findings of various preclinical SAH models,²⁴ a potential mechanism underlying hydrocephalus is suggested to be the CSF hypersecretion from the choroid plexus. In the rabbit cisternal blood injection SAH model, Aydin et al.¹⁰⁰ revealed a large number of water-filled vesicles within the choroid plexus at 2–14 days after SAH, supporting the theory of choroid plexus hypersecretion. They also proposed that the increased number of cytoplasmic water vesicles is probably induced by the stimulation of the glossopharyngeal and vagus nerves, which innervate the choroid-related arteries. The second contributor may be the dysfunction of channels and transporters on choroid plexus epithelium and ependyma after SAH. The expression of water channels, AQP1 and AQP5, on the apical and basolateral membranes were increased 1–7 days at the protein and mRNA levels after IVH in the preterm rabbit,¹⁰¹ whereas the AQP4 found on the ependymal cells was increased at 1–3 days after SAH in rats.¹⁰² It should be mentioned that both AQP1 and AQP4 levels highly correlated with the hydrocephalus occurrence.¹⁰² Besides, the apical membrane potassium cotransporter, NKCC1, was also reportedly upregulated 1–7 days after IVH in rats, and was accompanied with the CSF secretion.¹⁰³ Thirdly, inflammatory responses and choroid plexus damage were linked. The epithelia can increase their rate of fluid secretion in response to proinflammatory stimuli, such as fibrin, DAMPs, hemoglobin, and heme.¹⁰³ IVH reportedly activates Toll-like receptor 4 (TLR4)- and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-dependent inflammatory response in the choroid plexus epithelium and the downstream Ste20-type stress kinase (SPAK), which stimulates NKCC1 upregulation.¹⁰³ The peripheral immune cells are reportedly trafficked into the choroid plexus after SAH stimulation and are accompanied by increased levels of chemoattractant molecules CCL2, tumor necrosis factor-α (TNF-α), and interleukins.¹⁰⁴ The increased ICP and alteration of TJ proteins at the choroid plexus after SAH are also believed to contribute to the acute choroid plexus damage-induced CSF hypersecretion.^103–105

Several questions exist in the current study of choroid plexus pathophysiological changes after SAH. 1) Long-term choroid plexus degeneration was found to occur after SAH. However, its effect on actual CSF production remains questionable, as well as whether it is related to the chronic hydrocephalus;⁹⁷ 2) The choroid plexus epithelium forms the blood-CSF barrier and maintains the osmotic and hydrostatic gradients, but current knowledge pertaining to the acute and chronic changes after SAH on such barriers and gradients remains limited. 3) the specific liquid flow mechanism still requires further investigation, which should include both bilateral channels/transporters and TJ proteins that were mentioned previously in the section of physiological CSF dynamics.

Brain parenchymal system responses to SAH

Endothelium impairment initiates immediately after aneurysm rupture, which disrupts the local equilibrium existing between blood and CSF/ISF. The blood content flowing into the CSF/ISF increases the osmotic concentration, further driving water influx to the parenchymal side, and contributing to acute cerebral edema formation.¹⁶ Such dysfunction is associated with increased ICP and expansion of brain volume within 24 h after SAH in humans.¹⁰⁶

During the sub-acute and delayed phases (2–14d) after SAH, there is a decrease in TJ proteins, ion/water transporter dysfunction, matrix metalloproteinase (MMPs)-induced basal lamina degradation, neuroinflammation, and endothelial cell death.¹⁶ The brain suffers a global ischemia after the spread of blood from the local site, which makes the second phase of endothelium dysfunction partially similar to the ischemia-induced endothelial cell injury. The increased endothelial permeability peaked at 72 h after SAH in the rat endovascular perforation model.¹⁰⁷ Endothelial cell-related ionic and vasogenic edema successively contribute to the increased CSF production and global cerebral edema formation in this period after SAH.¹⁷ The dysfunction of ion and water transporters on astrocytes resulted in Na⁺ influx into the intracellular space with low osmotic concentrations in the interstitial space. The changes of the Na⁺ gradient between the vascular compartment and interstitial compartment drives the subsequent influx of ionic edema fluid from the vascular compartment to the interstitial compartment.¹⁰⁸ This transmembrane ion (Na⁺) transport relies on endothelial channels and transporters, such as NHE,¹⁰⁹ NKCC1,¹¹⁰ and sulfonylurea receptor (SUR).¹¹¹ The Cl^– and water crosses the endothelium accompanied with Na⁺, and further improves CSF production. Furthermore, with the disruption of TJs and with the death of endothelial cells, the brain capillaries form the permeability pores, which allow plasma proteins, such as albumin and IgG, to diffuse into the brain interstitial compartment.¹¹² Hydrostatic and osmotic pressure gradients directly prompt further water influx.¹¹³

CSF absorption responses to SAH

Arachnoid villi responses to SAH

Blockage of arachnoid villi CSF drainage is initiated during the acute phase, and is sustained until the late stage after SAH. A brain autopsy revealed that the accumulation of blood clots in arachnoid granulations inhibited CSF drainage, raising the ICP after SAH in patients.¹¹⁴ This granulation blockage could still be observed under the microscope at 4 weeks after SAH.¹¹⁴ In addition to the blood clot, collagen and fibrin deposition were also found to block the arachnoid granulation-mediated CSF drainage in the autopsy.^84,115 Proliferation of leptomeningeal cells with increased collagen synthesis enzyme (prolyl 4-hydroxylase) prompts CSF collagen production in response to the circulating toxic products of blood, which further block the arachnoid granulations.^84,116 After SAH, the physiological structure of arachnoid granulations is destroyed by the increased ICP and by the chemical effects of digestive enzymes released from inflammatory cells,⁸⁴ which consequently stimulates the fibro-cells to facilitate granulation fibrosis.¹¹⁵ Fibrin/fibrinogen derived from the impaired BBB could be an additional source of granulation fibrin deposition.¹¹⁷ Knowledge regarding post-SAH pathophysiological changes of arachnoid villi has stagnated, as most studies were performed 20 years ago. A recent in vitro study demonstrated the significant decrease in small molecule permeability through arachnoid membranes when treated with whole and lysed blood.¹¹⁸ Future studies should focus on other potential underlying mechanisms involving arachnoid granulation blockage, and they should uncover how different ICPs affect erythrocyte clearance by the arachnoid granulation after SAH.

Lymphatic drainage responses to SAH

The lymphatic drainage plays an important role in post-SAH CSF drainage. Emerging animal studies show that ligation of cervical lymphatic drainage worsens early brain injury (brain edema, oxidative stress, and neuronal apoptosis) within 72 hours after SAH.^119–121 Since the discovery of meningeal lymphatic vessels, it has been widely accepted that they are involved in clearing amyloid-β in mouse (both aged and young) models of Alzheimer’s disease.^60,122 The finding that the meningeal lymphatic drainage clears macromolecule waste in the CNS raises the question as to whether it also participates in post-SAH blood clearance. Interestingly, a recent study revealed that the erythrocytes in CSF drained into the deep cervical lymph nodes via meningeal lymphatic vessels in mice 4 hours after SAH was induced by cisterna magna autologous blood injection.⁸⁵ Ablation of meningeal lymphatic vessels blocked erythrocytic drainage into the deep cervical lymph nodes, and aggravated neuroinflammation and short-term neurological deficits.⁸⁵ Meningeal lymphatic flow was augmented for 7 days after SAH without lymphangiogenesis or expansion.⁸⁵ The augmented meningeal lymphatic flow may be in response to the increased CSF production and neuroinflammation after SAH, similar to the functionality of the peripheral lymphatic system.^123,124 The inhibition of lymphatic growth factor, VEGFR3, exacerbated the neuroinflammation and neurobehavioral impairments 30 days after SAH,⁸⁵ suggesting the presence of slow lymphangiogenesis in the chronic phase.

Future studies are necessary to elucidate whether erythrocytes and their products of degradation enter meningeal lymphatics vessel through glymphatic pathways, and to determine how meningeal lymphatics regulate the immune response to SAH. An improved understanding of the mechanisms and regulation of lymphatic drainage in both the acute and chronic phases following SAH would be meaningful.

CSF movement and glymphatic system responses to SAH

The ventricular CSF movement is significantly blocked after the blood coagulation response is initiated in the subarachnoid space and ventricular system after SAH. And it would be life-threatening when the blood clot blocks the third and fourth ventricles, which may lead to severe hydrocephalus and herniation.¹²⁵

Meanwhile, decreased glymphatic fluid exchanges were recently observed in rodents and non-human primates after SAH.^17,125–129 Gaberel et al.¹²⁵ first demonstrated the impaired glymphatic system after SAH by using MRI in the rat SAH model of chiasmatic cistern blood injection. The diffusion of contrast agent, DOTA-Gd (557.6 Da), from the cisterna magna into parenchymal and olfactory bulbs was significantly restricted in the acute phase (24 h) after SAH.¹²⁵ Further research found that this impairment started as soon as SAH was induced in non-human primates.¹²⁷ In addition, the impaired distribution of both low molecular tracer fluorescent microspheres (0.02 μm) and large molecular tracers, such as Evans Blue (961 Da) and fluorophore Alex594 (758 Da) from cisterna magna to perivascular space and parenchyma, were also blocked 24 hours after SAH in rodents.^17,129 Consistently, we demonstrated that the clearance speeds of Evans blue from the cisterna magna to deep cervical lymph nodes and blood were simultaneously decreased at 24 hours after arterial perforation-induced SAH in rats.¹⁷

The blockage of glymphatic fluid exchange is attributed to several pathological changes after SAH. The primary cause is considered the occlusion and stimulation of the perivascular space by blood components.⁵ Blood rapidly flows into the perivascular space following SAH and diffuses into the perivascular parenchyma, resulting in a sequelae of pathological processes, including cerebral vasospasm, cytotoxic edema, blood coagulation in CSF, and other early brain injury.^17,126 Cerebral vasospasm enlarges the perivascular spaces and doubles glymphatic inflow speeds, facilitating immediate cytotoxic edema.¹³⁰ With the initiation of CSF blood coagulation, accumulating fibrin/fibrinogen deposits occupy the perivascular space and inhibit CSF-ISF exchange.^125,129 The fibrin/fibrinogen was found to exist in CSF for 7 days, even in the absence of a visible blood clot.^117,128,129 Hypoxia, cytokine infiltration, and blood toxicity prompts AQP4 depolarization on astrocytes in the first 24 hours after SAH, which is considered the mechanism of cytotoxic edema formation.^17,128 However, AQP4 knockout did not improve brain water content or neurological deficits.^126,131 Generally, AQP4 on the astrocytic endfeet facilitate CSF/ISF fluid exchange from the arterial side to the venous side. After brain injury, dislocated AQP4 impaired glymphatic function, resulting in water accumulation in astrocytes and brain parenchyma.⁵ The potential mechanism of AQP4 depolarization was attributed to the activation of SUR1/TRPM4 on the membranes of astrocyte bodies, which recruit AQP4 to form a heteromultimeric complex and cytotoxic edema.¹¹¹ Additionally, the expression of other transporters (e.g. NKCC1, excitatory amino acid transporter, glucose transporter) were increased in astrocytic cell somas, and contributed to the hypoxia-induced cytotoxic edema, which could be the potential cause of AQP4 depolarization.¹⁰⁸ SUR1 is only expressed during ATP deficiency, which may not last to the late stage of SAH.¹¹¹ However, the AQP4 depolarization reportedly lasts from days to a month after cerebral hemorrhage in rodents.^126,132 The long-term glymphatic changes warrant further exploration. Other transporters (e.g. NKCC1, excitatory amino acid transporter, and glucose transporter) increased expression on the astrocytic cell soma and contributed to the cytotoxic edema after hypoxia induction, which could also be the potential cause of AQP4 depolarization.¹⁰⁸

Therapeutic targets of CSF dynamic modulation after SAH

Surgical interventions, such as lumbar cistern drainage, external ventricular drainage, ventriculoperitoneal shunt, and endoscopic third ventriculostomy, are widely used in the clinical setting to accelerate blood CSF clearance and improve clinical outcome in SAH patients.¹⁴ Of note, pharmacological interventions may also be a potential treatment to assist in post-SAH clearance of blood CSF. Here, we focus on the molecular therapeutic targets of CSF dynamic modulation after SAH.

Intracisternal fibrinolysis is considered a promising treatment in favor of reducing subarachnoid clot and fibrin deposition in CSF for an extended period after SAH. Several randomized trials have shown that intracisternal administration of tissue plasminogen activator (tPA) and urokinase improved neurological outcomes in SAH patients, including the prevention of cerebral vasospasm,¹³³ delayed cerebral ischemia,¹³⁴ and hydrocephalus.¹³⁵ As mentioned earlier, subarachnoid clot and fibrin deposition could block CSF/ISF exchange and drainage via arachnoid villi. A recent pre-clinical study demonstrated that intraventricular injection of tPA cleared the perivascular space fibrin deposition and promoted glymphatic function after SAH in mice.^125,129 Fibrin deposition in the subarachnoid space mainly results from the activation of the extrinsic coagulation cascades mediated by glycoprotein tissue factor (TF) on the cell membrane surface.^129,136 The brain expresses high levels of TF on astrocytes.¹³⁷ One study showed that neutralized TF in CSF could also reduce fibrin deposition and improve glymphatic function.¹²⁹ However, the causes of post-SAH brain TF activation remain unclear. TF was a link between inflammation and coagulation in the peripheral system.¹³⁸ Thus, TF may serve as a promising target of anti-fibrin deposition and anti-neuroinflammation after SAH.

Water/ion transporters and channels are another potential molecular target for modulating CSF dynamics after SAH. Since AQP1 and NKCC1 have an important role in choroid plexus injury-induced CSF hypersecretion and hydrocephalus after SAH,^102,103 it is rational to evaluate the efficacy of their inhibitors in reducing CSF production. A variety of endothelial water/ion transporters and channels mediate endothelium (BBB)-derived CSF hypersecretion. The inhibition of SUR1/TRPM4 by glibenclamide alleviated BBB disruption and brain edema.¹³⁹ Additionally, selective metabotropic glutamate receptor 1 (mGluR1), a negative allosteric modulator, prevented BBB disruption and edema formation after SAH.¹⁴⁰ We recently demonstrated that pituitary adenylate cyclase-activating peptide (PACAP) provided potent BBB and glymphatic protection by down-regulating SUR1 expression on endothelial cells and astrocytes, attenuating the post-SAH astrocytic AQP4 translocation to the soma, promoting CSF movement in the subarachnoid and perivascular spaces, and accelerating CSF clearance to the deep cervical lymph nodes and blood.¹⁷ Thus, a better understanding of the function of other transporters and channels (e.g. NKCCs, NHE) located on endothelial cells (BBB), astrocytes (glymphatic function), and the choroid plexus after SAH would further facilitate the development of targeted pharmacological therapeutics for SAH patients.

Furthermore, the inflammatory response and oxidative stress may also play critical roles in dysregulating CSF dynamics after SAH.¹⁴¹ Inhibition of oxidative stress and inflammatory responses in the acute phase after SAH could prevent CSF hypersecretion and dysfunction of endothelial cells and choroid plexus dysfunction.^142,143 A recent study reported that lipopolysaccharide (LPS)-induced inflammation markedly limited perivascular CSF tracer flow and penetration into the parenchyma of mice. However, no AQP4 depolarization was found in their study. They attributed this glymphatic system dysfunction to TLR4 activation-induced choroid plexus hypersecretion.¹⁴⁴ It is insufficient evidence to support the proposal that chronic plexus hypersecretion may cause glymphatic system dysfunction. LPS is considered a potent pyroptosis inducer for immune cells, including astrocytes.¹⁴⁵ Astrocytes have large amount TF and fibrinogen in brain,¹³⁷ and we propose that LPS may induce astrocytic pyropotsis and TF release and fibrin formation, which blocks the perivascular space. However, it is known that the expression of glymphatic system key channel protein, AQP4, is widely affected by cytokines and reactive oxygen species,¹⁴⁶ but the effects on the AQP4 polarization remains unknown. In addition, a recent study revealed that meningeal and perivascular macrophages are involved in erythrocyte uptake at 2–5 days after SAH in mice, which highlights the importance of the glymphatic-lymphatic-mediated immune response to the post-SAH outcome.¹⁴⁷ The interventions that target the inflammatory response and oxidative stress, with benefits to post-SAH CSF circulation in the glymphatic-lymphatic system, are worthy of further investigation.

Conclusion

The new findings of the glymphatic system and meningeal lymphatics reshape our traditional knowledge that the choroid plexus–ventricle-arachnoid villi system is the sole pathway of CSF circulation. Undoubtedly, both systems work as an entire network to regulate CSF dynamics. However, due to a lack of real-time, high-quality methodology for monitoring fluid exchange between blood, CSF, ISF, meningeal lymphatics and extracranial lymphatic network, the concept of a newly proposed glymphatic- meningeal lymphatics system remains debatable. Besides, the proportions by which each system is responsible for CSF production and drainage remains unclear. It also remains unknown how specific proteins (e.g. ions/water transporters and channels) and what mechanisms (e.g. hydrostatic and osmotic driven) regulate the movement of ions, water, and immune cells in CSF. Overall, uncovering the participation of the glymphatic-meningeal lymphatic system in SAH will introduce a new modality for using CSF modulation as treatment for SAH. However, current research of CSF dynamics in the setting of SAH remains limited to investigations of visible CSF movement changes and blood metabolites after SAH insults, rather than molecular and cellular mechanism. Altogether, a better and deeper understanding of the dynamics of CSF physiology could provide a solid foundation for identifying the pathophysiological changes of post-SAH CSF dynamics. The functionality clarification of specific proteins that participate in fluid exchange in the CSF circulatory network at molecular and cellular levels would further assist in identifying therapeutic targets against blood clearance, cerebral edema formation, and hydrocephalus after SAH. Moreover, future studies are also needed to explore the relationship between other risk factors (e.g. hypoxia, oxidative stress, and inflammation) and pathophysiological dynamics of CSF circulation.

Footnotes

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This review was supported by grants from the National Institutes of Health (NS081740 and NS082184) of John H. Zhang, and the National Natural Science Foundation of China (82071287 and 81870916) of Jianmin Zhang, (81971107) of Sheng Chen

Acknowledgements

All figures were created using Biorender.com.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

ORCID iDs

Cameron Lenahan

Sheng Chen

References

Herbowski

The maze of the cerebrospinal fluid discovery. Anat Res Int 2013; 2013: 596027–592014.

Brinker

Stopa

Morrison

, et al. A new look at cerebrospinal fluid circulation. Fluids Barriers CNS 2014; 11: 10.

Johanson

Duncan

3rd Klinge

, et al. Multiplicity of cerebrospinal fluid functions: New challenges in health and disease. Cerebrospinal Fluid Res 2008; 5: 10.

Louveau

Smirnov

Keyes

, et al. Structural and functional features of central nervous system lymphatic vessels. Nature 2015; 523: 337–341.

Rasmussen

Mestre

Nedergaard

The glymphatic pathway in neurological disorders. Lancet Neurol 2018; 17: 1016–1024.

Jessen

Munk

Lundgaard

, et al. The glymphatic system: a beginner's guide. Neurochem Res 2015; 40: 2583–2599.

Wright

Lai

Sinclair

AJ.

Cerebrospinal fluid and lumbar puncture: a practical review. J Neurol 2012; 259: 1530–1545.

Sakka

Coll

Chazal

Anatomy and physiology of cerebrospinal fluid. Eur Ann Otorhinolaryngol Head Neck Dis 2011; 128: 309–316.

Bothwell

Janigro

Patabendige

Cerebrospinal fluid dynamics and intracranial pressure elevation in neurological diseases. Fluids Barriers CNS 2019; 16: 9.

10.

Plog

Nedergaard

The glymphatic system in Central nervous system health and disease: past, present, and future. Annu Rev Pathol 2018; 13: 379–394.

11.

Abbott

Pizzo

Preston

, et al. The role of brain barriers in fluid movement in the CNS: is there a ‘glymphatic' system? Acta Neuropathol 2018; 135: 387–407.

12.

Lovelock

Rinkel

Rothwell

PM.

Time trends in outcome of subarachnoid hemorrhage: population-based study and systematic review. Neurology 2010; 74: 1494–1501.

13.

Nieuwkamp

Setz

Algra

, et al. Changes in case fatality of aneurysmal subarachnoid haemorrhage over time, according to age, sex, and region: a meta-analysis. Lancet Neurol 2009; 8: 635–642.

14.

Macdonald

Schweizer

TA.

Spontaneous subarachnoid haemorrhage. Lancet 2017; 389: 655–666.

15.

Fujii

Yan

Rolland

, et al. Early brain injury, an evolving frontier in subarachnoid hemorrhage research. Transl Stroke Res 2013; 4: 432–446.

16.

Tso

Macdonald

RL.

Subarachnoid hemorrhage: a review of experimental studies on the microcirculation and the neurovascular unit. Transl Stroke Res 2014; 5: 174–189.

17.

Fang

Shi

Ren

, et al. Pituitary adenylate cyclase-activating polypeptide attenuates brain edema by protecting blood-brain barrier and glymphatic system after subarachnoid hemorrhage in rats. Neurotherapeutics 2020; 17: 1954–1972.

18.

Zhang

Liu

Fan

, et al. The GluN1/GluN2B NMDA receptor and metabotropic glutamate receptor 1 negative allosteric modulator has enhanced neuroprotection in a rat subarachnoid hemorrhage model. Exp Neurol 2018; 301: 13–25.

19.

Fumoto

Naraoka

Katagai

, et al. The role of oxidative stress in microvascular disturbances after experimental subarachnoid hemorrhage. Transl Stroke Res 2019; 10: 684–694.

20.

Coulibaly

Provencio

JJ.

Aneurysmal subarachnoid hemorrhage: an overview of inflammation-induced cellular changes. Neurotherapeutics 2020; 17: 436–445.

21.

Staykov

Schwab

Clearing bloody cerebrospinal fluid: clot lysis, neuroendoscopy and lumbar drainage. Curr Opin Crit Care 2013; 19: 92–100.

22.

Fang

Shao

, et al. The effectiveness of lumbar cerebrospinal fluid drainage in aneurysmal subarachnoid hemorrhage with different bleeding amounts. Neurosurg Rev 2020; 43: 739–747.

23.

Damkier

Brown

Praetorius

Cerebrospinal fluid secretion by the choroid plexus. Physiol Rev 2013; 93: 1847–1892.

24.

Solar

Zamani

Kubickova

, et al. Choroid plexus and the blood-cerebrospinal fluid barrier in disease. Fluids Barriers CNS 2020; 17: 35.

25.

Abbott

NJ.

Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology. Neurochem Int 2004; 45: 545–552.

26.

Praetorius

Nejsum

Nielsen

A SCL4A10 gene product maps selectively to the basolateral plasma membrane of choroid plexus epithelial cells. Am J Physiol Cell Physiol 2004; 286: C601–610.

27.

Hladky

Barrand

MA.

Fluid and ion transfer across the blood-brain and blood-cerebrospinal fluid barriers: a comparative account of mechanisms and roles. Fluids Barriers CNS 2016; 13: 19–11.

28.

Boron

WF.

Evaluating the role of carbonic anhydrases in the transport of HCO_3-related species. Biochim Biophys Acta 2010; 1804: 410–421.

29.

Alper

SL.

Molecular physiology and genetics of Na+-independent SLC₄ anion exchangers. J Exp Biol 2009; 212: 1672–1683.

30.

Praetorius

Damkier

HH.

Transport across the choroid plexus epithelium. Am J Physiol Cell Physiol 2017; 312: C673–C686.

31.

Keep

Xiang

Betz

AL.

Potassium cotransport at the rat choroid plexus. Am J Physiol 1994; 267: C1616–1622.

32.

Kanaka

Ohno

Okabe

, et al. The differential expression patterns of messenger RNAs encoding K-Cl cotransporters (KCC1,2) and Na-K-2Cl cotransporter (NKCC1) in the rat nervous system. Neuroscience 2001; 104: 933–946.

33.

Zeuthen

Cotransport of K+, Cl- and H₂O by membrane proteins from choroid plexus epithelium of necturus maculosus. J Physiol 1994; 478 (Pt 2): 203–219.

34.

Roepke

Kanda

Purtell

, et al. KCNE2 forms potassium channels with KCNA3 and KCNQ1 in the choroid plexus epithelium. Faseb J 2011; 25: 4264–4273.

35.

Spector

Keep

Robert Snodgrass

, et al. A balanced view of choroid plexus structure and function: focus on adult humans. Exp Neurol 2015; 267: 78–86.

36.

Krug

Gunzel

Conrad

, et al. Charge-selective claudin channels. Ann N Y Acad Sci 2012; 1257: 20–28.

37.

Abbott

Patabendige

Dolman

, et al. Structure and function of the blood-brain barrier. Neurobiol Dis 2010; 37: 13–25.

38.

Aird

Becker

RA.

The blood-brain barrier in clinical disease: a review. J Nerv Ment Dis 1963; 136: 517–526.

39.

Ronaldson

Davis

TP.

Regulation of blood-brain barrier integrity by microglia in health and disease: a therapeutic opportunity. J Cereb Blood Flow Metab 2020; 40: S6–S24.

40.

EH.

Leaky memories: impact of APOE4 on blood-brain barrier and dementia. J Cereb Blood Flow Metab 2020; 40: 1912–1914.

41.

Oreskovic

Klarica

The formation of cerebrospinal fluid: nearly a hundred years of interpretations and misinterpretations. Brain Res Rev 2010; 64: 241–262.

42.

Petrova

Koh

GY.

Biological functions of lymphatic vessels. Science 2020; 369(6500): eaax4063. DOI: 10.1126/science.aax4063.

43.

Pollay

The function and structure of the cerebrospinal fluid outflow system. Cerebrospinal Fluid Res 2010; 7: 9–06.

44.

Tripathi

RC.

The functional morphology of the outflow systems of ocular and cerebrospinal fluids. Exp Eye Res 1977; 25 Suppl: 65–116.

45.

Weed

LH.

Studies on Cerebro-Spinal fluid. No. III: the pathways of escape from the subarachnoid spaces with particular reference to the arachnoid villi. J Med Res. 1914; 31: 51–91.

46.

Welch

Pollay

Perfusion of particles through arachnoid villi of the monkey. Am J Physiol 1961; 201: 651–654.

47.

Grzybowski

Holman

Katz

, et al. In vitro model of cerebrospinal fluid outflow through human arachnoid granulations. Invest Ophthalmol Vis Sci 2006; 47: 3664–3672.

48.

Courtice

Simmonds

WJ.

The removal of protein from the subarachnoid space. Aust J Exp Biol Med Sci 1951; 29: 255–263.

49.

Boulton

Flessner

Armstrong

, et al. Determination of volumetric cerebrospinal fluid absorption into extracranial lymphatics in sheep. Am J Physiol 1998; 274: R88–96.

50.

Bradbury

Cserr

Westrop

RJ.

Drainage of cerebral interstitial fluid into deep cervical lymph of the rabbit. Am J Physiol 1981; 240: F329–336.

51.

Luedemann

Kondziella

Tienken

, et al. Spinal cerebrospinal fluid pathways and their significance for the compensation of kaolin-hydrocephalus. Acta Neurochir Suppl 2002; 81: 271–273.

52.

Johnston

Zakharov

Papaiconomou

, et al. Evidence of connections between cerebrospinal fluid and nasal lymphatic vessels in humans, non-human primates and other mammalian species. Cerebrospinal Fluid Res 2004; 1: 2–2005.

53.

Ahn

Cho

Kim

, et al. Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid. Nature 2019; 572: 62–66.

54.

Bradbury

Westrop

RJ.

Factors influencing exit of substances from cerebrospinal fluid into deep cervical lymph of the rabbit. J Physiol 1983; 339: 519–534.

55.

Nagra

Koh

Zakharov

, et al. Quantification of cerebrospinal fluid transport across the cribriform plate into lymphatics in rats. Am J Physiol Regul Integr Comp Physiol 2006; 291: R1383–1389.

56.

Zakharov

Papaiconomou

Koh

, et al. Integrating the roles of extracranial lymphatics and intracranial veins in cerebrospinal fluid absorption in sheep. Microvasc Res 2004; 67: 96–104.

57.

Brinker

Ludemann

Berens von Rautenfeld

, et al. Dynamic properties of lymphatic pathways for the absorption of cerebrospinal fluid. Acta Neuropathol 1997; 94: 493–498.

58.

Aspelund

Antila

Proulx

, et al. A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med 2015; 212: 991–999.

59.

Ineichen

Detmar

, et al. Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice. Nat Commun 2017; 8: 1434.

60.

Da Mesquita

Louveau

Vaccari

, et al. Functional aspects of meningeal lymphatics in ageing and alzheimer's disease. Nature 2018; 560: 185–191.

61.

Absinta

Nair

, et al. Human and nonhuman primate meninges harbor lymphatic vessels that can be visualized noninvasively by MRI. Elife 2017; 6. DOI: 10.7554/elife.29738.

62.

Louveau

Herz

Alme

, et al. CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature. Nat Neurosci 2018; 21: 1380–1391.

63.

Antila

Karaman

Nurmi

, et al. Development and plasticity of meningeal lymphatic vessels. J Exp Med 2017; 214: 3645–3667.

64.

Da Mesquita

Herz

Wall

, et al. Aging-associated deficit in CCR7 is linked to worsened glymphatic function, cognition, neuroinflammation, and beta-amyloid pathology. Sci Adv 2021; 7(21): eabe4601. DOI: 10.1126/sciadv.abe4601.

65.

Da Mesquita

Papadopoulos

Dykstra

, Dominantly Inherited Alzheimer Networket al. Meningeal lymphatics affect microglia responses and anti-Abeta immunotherapy. Nature 2021; 593: 255–260.

66.

Nedergaard

Neuroscience. Garbage truck of the brain. Science 2013; 340: 1529–1530.

67.

Iliff

Wang

Liao

, et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci Transl Med 2012; 4: 147ra111.

68.

Bechet

Kylkilahti

Mattsson

, et al. Light sheet fluorescence microscopy of optically cleared brains for studying the glymphatic system. J Cereb Blood Flow Metab 2020; 40: 1975–1986.

69.

Xie

Kang

, et al. Sleep drives metabolite clearance from the adult brain. Science 2013; 342: 373–377.

70.

Lee

Xie

, et al. The effect of body posture on brain glymphatic transport. J Neurosci 2015; 35: 11034–11044.

71.

van Veluw

Hou

Calvo-Rodriguez

, et al. Vasomotion as a driving force for paravascular clearance in the awake mouse brain. Neuron 2020; 105: 549–561.

72.

Iliff

Wang

Zeppenfeld

, et al. Cerebral arterial pulsation drives paravascular CSF-interstitial fluid exchange in the murine brain. J Neurosci 2013; 33: 18190–18199.

73.

Goodman

Iliff

JJ.

Vasomotor influences on glymphatic-lymphatic coupling and solute trafficking in the Central nervous system. J Cereb Blood Flow Metab 2020; 40: 1724–1734.

74.

Pizzo

Wolak

Kumar

, et al. Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport and osmotic enhancement of delivery. J Physiol 2018; 596: 445–475.

75.

Iliff

Lee

, et al. Brain-wide pathway for waste clearance captured by contrast-enhanced MRI. J Clin Invest 2013; 123: 1299–1309.

76.

Mathiisen

Lehre

Danbolt

, et al. The perivascular astroglial sheath provides a complete covering of the brain microvessels: an electron microscopic 3D reconstruction. Glia 2010; 58: 1094–1103.

77.

Lundgaard

Yang

, et al. Glymphatic clearance controls state-dependent changes in brain lactate concentration. J Cereb Blood Flow Metab 2017; 37: 2112–2124.

78.

Cui

Wang

Zheng

, et al. Decreased AQP4 expression aggravates a-Synuclein pathology in Parkinson's disease mice, possibly via impaired glymphatic clearance. J Mol Neurosci. Epub ahead of print 26 March 2021. DOI: 10.1007/s12031-021-01836-4.

79.

Sehba

Pluta

Zhang

JH.

Metamorphosis of subarachnoid hemorrhage research: from delayed vasospasm to early brain injury. Mol Neurobiol 2011; 43: 27–40.

80.

Small

JM.

Diagnosis and management of subarachnoid haemorrhage. Trans Med Soc Lond 1970; 86: 25–26.

81.

Cahill

Calvert

Solaroglu

, et al. Vasospasm and p53-induced apoptosis in an experimental model of subarachnoid hemorrhage. Stroke 2006; 37: 1868–1874.

82.

Wan

Hua

Garton

HJL

, et al. Activation of epiplexus macrophages in hydrocephalus caused by subarachnoid hemorrhage and thrombin. CNS Neurosci Ther 2019; 25: 1134–1141.

83.

Chen S, Xu P, Fang Y, et al. The Updated Role of the Blood Brain Barrier in Subarachnoid Hemorrhage: From Basic and Clinical Studies. Curr Neuropharmacol 2020; 18: 1266–1278.

84.

Motohashi

Suzuki

Shida

, et al. Subarachnoid haemorrhage induced proliferation of leptomeningeal cells and deposition of extracellular matrices in the arachnoid granulations and subarachnoid space. Immunhistochemical study. Acta Neurochir (Wien) 1995; 136: 88–91.

85.

Chen

Wang

, et al. Meningeal lymphatics clear erythrocytes that arise from subarachnoid hemorrhage. Nat Commun 2020; 11: 3159.

86.

Rosen

Macdonald

Huo

, et al. Intraventricular hemorrhage from ruptured aneurysm: clinical characteristics, complications, and outcomes in a large, prospective, multicenter study population. J Neurosurg 2007; 107: 261–265.

87.

Jabbarli

Reinhard

Roelz

, et al. The predictors and clinical impact of intraventricular hemorrhage in patients with aneurysmal subarachnoid hemorrhage. Int J Stroke 2016; 11: 68–76.

88.

Xiang

Routhe

Wilkinson

, et al. The choroid plexus as a site of damage in hemorrhagic and ischemic stroke and its role in responding to injury. Fluids Barriers CNS 2017; 14: 8.

89.

Fukumizu

Takashima

Becker

LE.

Glial reaction in periventricular areas of the brainstem in fetal and neonatal posthemorrhagic hydrocephalus and congenital hydrocephalus. Brain Dev 1996; 18: 40–45.

90.

Fukumizu

Takashima

Becker

LE.

Neonatal posthemorrhagic hydrocephalus: neuropathologic and immunohistochemical studies. Pediatr Neurol 1995; 13: 230–234.

91.

Gram

Sveinsdottir

Cinthio

, et al. Extracellular hemoglobin – mediator of inflammation and cell death in the choroid plexus following preterm intraventricular hemorrhage. J Neuroinflammation 2014; 11: 200.

92.

Simard

Tosun

Melnichenko

, et al. Inflammation of the choroid plexus and ependymal layer of the ventricle following intraventricular hemorrhage. Transl Stroke Res 2011; 2: 227–231.

93.

Gao

Liu

Chen

, et al. Hydrocephalus after intraventricular hemorrhage: the role of thrombin. J Cereb Blood Flow Metab 2014; 34: 489–494.

94.

Liszczak

Black

Tzouras

, et al. Morphological changes of the basilar artery, ventricles, and choroid plexus after experimental SAH. J Neurosurg 1984; 61: 486–493.

95.

Mayfrank

Kim

Kissler

, et al. Morphological changes following experimental intraventricular haemorrhage and intraventricular fibrinolytic treatment with recombinant tissue plasminogen activator. Acta Neuropathol 2000; 100: 561–567.

96.

Yilmaz

Aydin

Kanat

, et al. The effect of choroidal artery vasospasm on choroid plexus injury in subarachnoid hemorrhage: experimental study. Turk Neurosurg 2011; 21: 477–482.

97.

Kotan

Aydin

Gundogdu

, et al. Parallel development of choroid plexus degeneration and meningeal inflammation in subarachnoid hemorrhage – experimental study. Adv Clin Exp Med 2014; 23: 699–704.

98.

Chen

Feng

Tan

, et al. Post-hemorrhagic hydrocephalus: Recent advances and new therapeutic insights. J Neurol Sci 2017; 375: 220–230.

99.

Darkwah Oppong

Gembruch

Herten

, et al. Intraventricular hemorrhage caused by subarachnoid hemorrhage: Does the severity matter? World Neurosurg 2018; 111: e693–e702.

100.

Aydin

Kanat

Turkmenoglu

, et al. Changes in number of water-filled vesicles of choroid plexus in early and late phase of experimental rabbit subarachnoid hemorrhage model: the role of petrous ganglion of glossopharyngeal nerve. Acta Neurochir (Wien) 2014; 156: 1311–1317.

101.

Sveinsdottir

Gram

Cinthio

, et al. Altered expression of aquaporin 1 and 5 in the choroid plexus following preterm intraventricular hemorrhage. Dev Neurosci 2014; 36: 542–551.

102.

Long

Huang

, et al. The dynamic expression of aquaporins 1 and 4 in rats with hydrocephalus induced by subarachnoid haemorrhage. Folia Neuropathol 2019; 57: 182–195.

103.

Karimy

Zhang

Kurland

, et al. Inflammation-dependent cerebrospinal fluid hypersecretion by the choroid plexus epithelium in posthemorrhagic hydrocephalus. Nat Med 2017; 23: 997–1003.

104.

Solar

Klusakova

Jancalek

, et al. Subarachnoid hemorrhage induces dynamic immune cell reactions in the choroid plexus. Front Cell Neurosci 2020; 14: 18.

105.

Shrestha

Paul

Pachter

JS.

Alterations in tight junction protein and IgG permeability accompany leukocyte extravasation across the choroid plexus during neuroinflammation. J Neuropathol Exp Neurol 2014; 73: 1047–1061.

106.

Leinonen

Vanninen

Rauramaa

Raised intracranial pressure and brain edema. Handb Clin Neurol 2017; 145: 25–37.

107.

Liang

, et al. Blood-brain barrier permeability change and regulation mechanism after subarachnoid hemorrhage. Metab Brain Dis 2015; 30: 597–603.

108.

Stokum

Gerzanich

Simard

JM.

Molecular pathophysiology of cerebral edema. J Cereb Blood Flow Metab 2016; 36: 513–538.

109.

Lam

Wise

O'Donnell

ME.

Cerebral microvascular endothelial cell Na/H exchange: evidence for the presence of NHE1 and NHE2 isoforms and regulation by arginine vasopressin. Am J Physiol Cell Physiol 2009; 297: C278–289.

110.

O'Donnell

Tran

Lam

, et al. Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat Middle cerebral artery occlusion model of stroke. J Cereb Blood Flow Metab 2004; 24: 1046–1056.

111.

Stokum

Kwon

Woo

, et al. SUR1-TRPM4 and AQP4 form a heteromultimeric complex that amplifies ion/water osmotic coupling and drives astrocyte swelling. Glia 2018; 66: 108–125.

112.

Vorbrodt

Lossinsky

Wisniewski

, et al. Ultrastructural observations on the transvascular route of protein removal in vasogenic brain edema. Acta Neuropathol 1985; 66: 265–273.

113.

Durward

Del Maestro

Amacher

, et al. The influence of systemic arterial pressure and intracranial pressure on the development of cerebral vasogenic edema. J Neurosurg 1983; 59: 803–809.

114.

Torvik

Bhatia

Murthy

VS.

Transitory block of the arachnoid granulations following subarachnoid haemorrhage. A postmortem study. Acta Neurochir (Wien) 1978; 41: 137–146.

115.

Chopard

Brancalhao

Miranda-Neto

, et al. Arachnoid granulation affected by subarachnoid hemorrhage. Arq Neuropsiquiatr 1993; 51: 452–456.

116.

Sajanti

Bjorkstrand

Finnila

, et al. Increase of collagen synthesis and deposition in the arachnoid and the dura following subarachnoid hemorrhage in the rat. Biochim Biophys Acta 1999; 1454: 209–216.

117.

Golanov

Sharpe

Regnier-Golanov

, et al. Fibrinogen chains intrinsic to the brain. Front Neurosci 2019; 13: 541.

118.

Hansen

Romanova

Janson

, et al. The effects of blood and blood products on the arachnoid cell. Exp Brain Res 2017; 235: 1749–1758.

119.

Sun

Xia

Wang

, et al. Effects of blockade of cerebral lymphatic drainage on regional cerebral blood flow and brain edema after subarachnoid hemorrhage. Clin Hemorheol Microcirc 2006; 34: 227–232.

120.

Sun

Xie

Yang

, et al. Blocking cerebral lymphatic drainage deteriorates cerebral oxidative injury in rats with subarachnoid hemorrhage. Acta Neurochir Suppl 2011; 110: 49–53.

121.

Sun

Jia

Wang

, et al. Cerebral lymphatic blockage aggravates apoptosis of hippocampal neurons induced by cerebrospinal fluid from experimental subarachnoid hemorrhage. Sheng Li Xue Bao 2009; 61: 317–323.

122.

Wen

Yang

Wang

, et al. Induced dural lymphangiogenesis facilities soluble amyloid-beta clearance from brain in a transgenic mouse model of alzheimer's disease. Neural Regen Res 2018; 13: 709–716.

123.

Zhou

Wood

Schwarz

, et al. Near-infrared lymphatic imaging demonstrates the dynamics of lymph flow and lymphangiogenesis during the acute versus chronic phases of arthritis in mice. Arthritis Rheum 2010; 62: 1881–1889.

124.

Narimatsu

Hattori

Koike

, et al. Corneal lymphangiogenesis ameliorates corneal inflammation and edema in late stage of bacterial keratitis. Sci Rep 2019; 9: 2984.

125.

Gaberel

Gakuba

Goulay

, et al. Impaired glymphatic perfusion after strokes revealed by contrast-enhanced MRI: a new target for fibrinolysis? Stroke 2014; 45: 3092–3096.

126.

Luo

Yao

, et al. Paravascular pathways contribute to vasculitis and neuroinflammation after subarachnoid hemorrhage independently of glymphatic control. Cell Death Dis 2016; 7: e2160.

127.

Goulay

Flament

Gauberti

, et al. Subarachnoid hemorrhage severely impairs brain parenchymal cerebrospinal fluid circulation in nonhuman primate. Stroke 2017; 48: 2301–2305.

128.

Zou

Feng

, et al. Persistent malfunction of glymphatic and meningeal lymphatic drainage in a mouse model of subarachnoid hemorrhage. Exp Neurobiol 2019; 28: 104–118.

129.

Golanov

Bovshik

Wong

, et al. Subarachnoid hemorrhage – induced block of cerebrospinal fluid flow: Role of brain coagulation factor III (tissue factor). J Cereb Blood Flow Metab 2018; 38: 793–808.

130.

Mestre

Sweeney

, et al. Cerebrospinal fluid influx drives acute ischemic tissue swelling. Science 2020; 367. DOI: 10.1126/science.aax7171.

131.

Liu

Sun

Zhang

, et al. Aquaporin4 knockout aggravates early brain injury following subarachnoid hemorrhage through impairment of the glymphatic system in rat brain. Acta Neurochir Suppl 2020; 127: 59–64.

132.

Ding

Zhang

, et al. Astrogliosis inhibition attenuates hydrocephalus by increasing cerebrospinal fluid reabsorption through the glymphatic system after germinal matrix hemorrhage. Exp Neurol 2019; 320: 113003.

133.

Yamamoto

Esaki

Nakao

, et al. Efficacy of low-dose tissue-plasminogen activator intracisternal administration for the prevention of cerebral vasospasm after subarachnoid hemorrhage. World Neurosurg 2010; 73: 675–682.

134.

Seifert

Stolke

Zimmermann

, et al. Prevention of delayed ischaemic deficits after aneurysmal subarachnoid haemorrhage by intrathecal bolus injection of tissue plasminogen activator (rTPA). a prospective study. Acta Neurochir (Wien) 1994; 128: 137–143.

135.

Ramakrishna

Sekhar

Ramanathan

, et al. Intraventricular tissue plasminogen activator for the prevention of vasospasm and hydrocephalus after aneurysmal subarachnoid hemorrhage. Neurosurgery 2010; 67: 110–117; discussion 117.

136.

Clarke

Suggs

Diwan

, et al. Microvascular platelet aggregation and thrombosis after subarachnoid hemorrhage: a review and synthesis. J Cereb Blood Flow Metab 2020; 40: 1565–1575.

137.

Eddleston

de la Torre

Oldstone

, et al. Astrocytes are the primary source of tissue factor in the murine Central nervous system. A role for astrocytes in cerebral hemostasis. J Clin Invest 1993; 92: 349–358.

138.

Witkowski

Landmesser

Rauch

Tissue factor as a link between inflammation and coagulation. Trends Cardiovasc Med 2016; 26: 297–303.

139.

Caffes

Kurland

Gerzanich

, et al. Glibenclamide for the treatment of ischemic and hemorrhagic stroke. Int J Mol Sci 2015; 16: 4973–4984.

140.

Zhang C, Jiang M, Wang WQ, et al. Selective mGluR1 Negative Allosteric Modulator Reduces Blood-Brain Barrier Permeability and Cerebral Edema After Experimental Subarachnoid Hemorrhage. Transl Stroke Res 2020; 11: 799–811.

141.

Saand

Xing

, et al. CSF lipocalin-2 increases early in subarachnoid hemorrhage are associated with neuroinflammation and unfavorable outcome. J Cereb Blood Flow Metab 2021; 271678X211012110.

142.

Khey

KMW

Huard

Mahmoud

SH.

Inflammatory pathways following subarachnoid hemorrhage. Cell Mol Neurobiol 2020; 40: 675–693.

143.

Chrissobolis

Miller

Drummond

, et al. Oxidative stress and endothelial dysfunction in cerebrovascular disease. Front Biosci (Landmark Ed) 2011; 16: 1733–1745.

144.

Manouchehrian

Ramos

Bachiller

, et al. Acute systemic LPS-exposure impairs perivascular CSF distribution in mice. J Neuroinflammation 2021; 18: 34.

145.

Sun

Zhao

, et al. Dexmedetomidine inhibits astrocyte pyroptosis and subsequently protects the brain in in vitro and in vivo models of sepsis. Cell Death Dis 2019; 10: 167.

146.

Vandebroek

Yasui

Regulation of AQP4 in the Central nervous system. Int J Mol Sci 2020; 21: 1603. DOI: 10.3390/ijms21051603.

147.

Wan

Brathwaite

, et al. Role of perivascular and meningeal macrophages in outcome following experimental subarachnoid hemorrhage. J Cereb Blood Flow Metab 2021; 41: 1842–1857.