Mechanisms to explain the reverse perivascular transport of solutes out of the brain

Experimental studies and observations in the human brain indicate that interstitial fluid and solutes, such as amyloid-beta (Abeta), are eliminated from grey matter of the brain along pericapillary and periarterial pathways. It is unclear, however, what constitutes the motive force for such transport within blood vessel walls, which is in the opposite direction to blood flow. In this paper the potential for global pressure differences to achieve such transport are considered. A mathematical model is constructed in order to test the hypothesis that perivascular drainage of interstitial fluid and solutes out of brain tissue is driven by pulsations of the blood vessel walls. Here it is assumed that drainage occurs through a thin layer between astrocytes and endothelial cells or between smooth muscle cells. The model suggests that, during each pulse cycle, there are periods when fluid and solutes are driven along perivascular spaces in the reverse direction to the flow of blood. It is shown that successful drainage may depend upon some attachment of solutes to the lining of the perivascular space, in order to produce a valve-like effect, although an alternative without this requirement is also postulated. Reduction in pulse amplitude, as in ageing cerebral vessels, would prolong the attachment time, encourage precipitation of Abeta peptides in vessel walls, and impair elimination of Abeta from the brain. These factors may play a role in the pathogenesis of cerebral amyloid angiopathy and in the accumulation of Abeta in the brain in Alzheimer's disease.     

Ultrastructure of the meninges at the site of penetration of veins through the dura mater,with particular reference to Pacchionian granulations

At the sites where a vein penetrates through the dura mater, two aspects deserve particular attention: (i) The delineation of the perivascular cleft, a space belonging to the interstitial cerebrospinal fluid (CSF) compartment, toward the interior hemal milieu of the dura mater. (ii) The relationship between the perivascular arachnoid layer and the subdural neurothelium at the point of vascular penetration. These problems were investigated in the rat and in two species of New-World monkeys (Cebus apella, Callitrix jacchus). Concerning the first aspect, tight appositions of meningeal cells to the vessel wall, the basal lamina of which is widened and enriched with microfibrils, prevent communication between the interstitial CSF in the perivascular cleft and the hemal milieu in the dura mater. With reference to the second aspect, the perivascular arachnoid cells are transformed into neurothelial cells at the point where they become exposed to the hemal milieu of the dura mater and subsequently continuous with the subdural neurothelium. Leptomeningeal protrusions encompassing outer CSF space can penetrate into the dura mater. These protrusions may expand and branch repeatedly, forming along the wall of the dural sinus Pacchionian granulations. At these sites, however, the structural integrity of the sinus wall and the Pacchionian granulation is not lost. Numerous vesiculations not only in the sinus and vascular walls, but also in the cellular arrays of the Pacchionian granulations or paravascular leptomeningeal protrusions indicate mechanisms of transcellular fluid transport. Moreover, the texture of the leptomeningeal protrusions favors an additional function of these structures as a "volume" buffer.     

Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Background

Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it’s expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation.

Methods

We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student’s t- test.

Results

We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2?>?apoE3?>?apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state.

Conclusions

Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.

     

Aquaporin-4 gene disruption in mice reduces brain swelling and mortality in pneumococcal meningitis

The astroglial water channel aquaporin-4 (AQP4) facilitates water movement into and out of brain parenchyma. To investigate the role of AQP4 in meningitis-induced brain edema, Streptococcus pneumoniae was injected into cerebrospinal fluid (CSF) in wild type and AQP4 null mice. AQP4-deficient mice had remarkably lower intracranial pressure (9 +/- 1 versus 25 +/- 5 cm H2O) and brain water accumulation (2 +/- 1 versus 9 +/- 1 microl) at 30 h, and improved survival (80 versus 0% survival) at 60 h, through comparable CSF bacterial and white cell counts. Meningitis produced marked astrocyte foot process swelling in wild type but not AQP4 null mice, and slowed diffusion of an inert macromolecule in brain extracellular space. AQP4 protein was strongly up-regulated in meningitis, resulting in a approximately 5-fold higher water permeability (P(f)) across the blood-brain barrier compared with non-infected wild type mice. Mathematical modeling using measured P(f) and CSF dynamics accurately simulated the elevated lower intracranial pressure and brain water produced by meningitis and predicted a beneficial effect of prevention of AQP4 upregulation. Our findings provide a novel molecular mechanism for the pathogenesis of brain edema in acute bacterial meningitis, and suggest that inhibition of AQP4 function or up-regulation may dramatically improve clinical outcome.     

The effects of variation in the arterial pulse waveform on perivascular flow

Cerebrospinal fluid (CSF) enters nervous tissues through perivascular spaces. Flow through these pathways is important for solute transport and to prevent fluid accumulation. Syringomyelia is commonly associated with subarachnoid space obstructions such as Chiari I malformation. However, the mechanism of development of these fluid-filled cavities is unclear. Studies have suggested that changes in the arterial and CSF pressures could alter normal perivascular flow. This study uses an idealised model of the perivascular space to investigate how variation in the arterial pulse influences fluid flow. The model used simulated subarachnoid pressures from healthy controls (N = 9), Chiari patients with (N = 7) and without (N = 8) syringomyelia. A parametric analysis was conducted to determine how features of the arterial pulse altered flow. The features of interest included: the timing and magnitude of the peak displacement, and the area under the curve in the phases of uptake and decline. A secondary aim was to determine if the previously observed differences between subject groups were sensitive to variation in the arterial pulse wave. The study demonstrated that the Chiari patients without a syrinx maintained a significantly higher level of perivascular inflow over a physiologically likely range of pulse wave shapes. The analysis also suggested that age-related changes in the arterial pulse (i.e. increased late systolic pulse amplitude and faster diastolic decay), could increase resistance to perivascular inflow affecting solute transport.     

Electrolyte- and fluid-spaces of rat brain in situ after infusion with dinitrophenol.

Chemical distribution measurements of radioactive sodium-thiosulfate (35S) and of the brain water indicate that infusion of 2.4-dinitrophenol into a carotid artery of rats caused a water uptake and fluid shifts from the extra- into the intracellular compartments in the central nervous system. The extracellular marker compound was administered to the brain via ventriculo-cisternal perfusion and intravenous injection yielding almost equal concentrations in plasma-water and perfusate. In order to prevent an active efflux of the label from the tissue, high concentrations were utilized in the perfusate to saturate potential outward transport mechanisms. The indicator space (based on total brain water) was 16% in controls and 12% in experimental animals when marker equilibrium had been attained, which is equivalent in reduction of the extracellular space of about 1/4. Intracellular water and Na+ rose after DNP, while K+ remained all but unchanged. The fluid shift into the intracellular compartment was found to relate closely with a cellular uptake of Na+. The Na+ concentration both in plasma and in the perfusion fluid leaving the ventricular system was consistently reduced in experimental animals. The K+ concentration was significantly elevated in the plasma of experimental animals but virtually unchanged in the cisternal effluate.     

Evidence for bulk flow of brain interstitial fluid: significance for physiology and pathology

This review surveys evidence for the flow of brain interstitial fluid (ISF) via preferential pathways through the brain, and its relation to cerebrospinal fluid (CSF). Studies over >100 years have raised several controversial points, not all of them resolved. Recent studies have usefully combined a histological and a mathematical approach. Taken together the evidence indicates an ISF bulk flow rate of 0.1-0.3 microl min(-1) g(-1) in rat brain along preferential pathways especially perivascular spaces and axon tracts. The main source of this fluid is likely to be the brain capillary endothelium, which has the necessary ion transporters, channels and water permeability to generate fluid at a low rate, c1/100th of the rate per square centimeter of CSF secretion across choroid plexus epithelium. There is also evidence that a proportion of CSF may recycle from the subarachnoid space into arterial perivascular spaces on the ventral surface of the brain, and join the circulating ISF, draining back via venous perivascular spaces and axon tracts into CSF compartments, and out both through arachnoid granulations and along cranial nerves to the lymphatics of the neck. The bulk flow of ISF has implications for non-synaptic cell:cell communication (volume transmission); for drug delivery, distribution, and clearance; for brain ionic homeostasis and its disturbance in brain edema; for the immune function of the brain; for the clearance of beta-amyloid deposits; and for the migration of cells (malignant cells, stem cells).     

Cerebrospinal fluid flow increases from newborn to adult stages

Solute transport through the brain is of major importance for the clearance of toxic molecules and metabolites, and it plays key roles in the pathophysiology of the central nervous system. This solute transport notably depends on the cerebrospinal fluid (CSF) flow, which circulates in the subarachnoid spaces, the ventricles and the perivascular spaces. We hypothesized that the CSF flow may be different in the perinatal period compared to the adult period. Using in vivo magnetic resonance imaging (MRI) and near‐infrared fluorescence imaging (NIRF), we assessed the dynamic of the CSF flow in rodents at different ages. By injecting a contrast agent into the CSF, we first used MRI to demonstrate that CSF flow gradually increases with age, with the adult pattern observed at P90. This observation was confirmed by NIRF, which revealed an increased CSF flow in P90 rats when compared with P4 rats not only at the surface of the brain but also deep in the brain structures. Lastly, we evaluated the exit routes of the CSF from the brain. We demonstrated that indocyanine green injected directly into the striatum spread throughout the parenchyma in adult rats, whereas it stayed at the injection point in P4 rats. Moreover, the ability of CSF to exit through the nasal mucosa was increased in the adult rodents. Our results provide evidence that the perinatal brain has nonoptimal CSF flow and exit and, thus, may have impaired clean‐up capacity. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018     

Uptake of 36Cl and 22Na by the Brain-Cerebrospinal Fluid System: Comparison of the Permeability of the Blood-Brain and Blood-Cerebrospinal Fluid Barriers

Abstract: Transport and permeability properties of the blood-brain and blood-CSF barriers were determined by kinetic analysis of radioisotope uptake from the plasma into the CNS of the adult rat. Cerebral cortex and cerebellum uptake curves for ³⁶Cl and ²²Na were resolved into two components. The fast component (t_½ 0.02–0.05 h, fractional volume 0.04–0.08) is comprised of the vascular compartment and a small perivascular space whereas the slow component (t_½ 1.06–1.69 h, fractional volume 0.92–0.96) represents isotope movement across the blood-brain barrier into the brain extracellular and cellular compartments. Uptake curves of both ³⁶Cl and ²²Na into the CSF were also resolved into two components, a fast component (t_½ 0.18 h, fractional volume 0.24) and a slow component (t_½ 1.2 h, fractional volume 0.76). Evidence suggests that the fast component represents isotope movement across the blood-CSF barrier, i.e., the choroid plexuses, whereas the CSF slow component probably reflects isotope penetration primarily from the brain extracellular fluid into the CSF. The extracellular fluid volume of the cerebral cortex and cerebellum was estimated as ?13% from the initial slope of the curve of brain space versus CSF space curve for both ³⁶Cl and ²²Na. Like the choroid plexuses, the glial cell compartment of the brain appears to accumulate Cl from 2 to 6 times that predicted for passive distribution. The relative permeability of the blood-CSF and blood-brain barriers to ³⁶Cl, ²²Na, and [³H]mannitol was determined by calculating permeability surface-area products (PA). Analysis of the PA values for all three isotopes indicates that the effective permeability of the choroidal epithelium (blood/CSF barrier) is significantly greater than that of the capillary endothelium in the cerebral cortex and cerebellum (blood-brain barrier).     

Electrolyte- and fluid-spaces of rat brain in situ after infusion with dinitrophenol

Chemical distribution measurements of radioactive sodium-thiosulfate (³⁵S) and of the brain water indicate that infusion of 2.4-dinitrophenol into a carotid artery of rats caused a water uptake and fluid shifts from the extra- into the intracellular compartments in the central nervous system. The extracellular marker compound was administered to the brain via ventriculo-cisternal perfusion and intravenous injection yielding almost equal concentrations in plasma- water and perfusate. In order to prevent an active efflux of the label from the tissue, high concentrations were utilized in the perfusate to saturate potential outward transport mechanisms. The indicator space (based on total brain water) was 16% in controls and 12% in experimental animals when marker equilibrium had been attained, which is equivalent in reduction of the extracellular space of about 1/4. Intracellular water and Na⁺ rose after DNP, while K⁺ remained all but unchanged. The fluid shift into the intracellular compartment was found to relate closely with a cellular uptake of Na⁺. The Na⁺ concentration both in plasma and in the perfusion fluid leaving the ventricular system was consistently reduced in experimental animals. The K⁺ concentration was significantly elevated in the plasma of experimental animals but virtually unchanged in the cisternal effluate.     

Permeable Endothelium and the Interstitial Space of Brain

1. Fenestrated vessels can be reversibly induced in brain by agents that stimulate urokinase production. This plasminogen activator, like vascular endothelial growth factor and metalloproteinases, is secreted by tumor cells and may account for induction of fenestrated vessels. Why only some of the brain's barrier vessels are converted to fenestrated vessels is unknown.2. The structures responsible for the filtering of solutes by fenestrated vessels may be the same as those of continuous, less permeable vessels: the glycocalyx on the surfaces of the endothelial cells and the subendothelial basal lamina.3. Solutes leaving the cerebral ventricles immediately enter the interstitial clefts between the cells lining the ventricles. A fraction of a variety of solutes, injected into CSF compartments, is retained by subendothelial basal lamina, from which the solutes may be released in a regulated way.4. The brain's CSF and interstitial clefts are the conduits for nonsynaptic volume transmission of diffusible signals, e.g., ions, neurotransmitters, and hormones. This type of transmission could be abetted by a parallel, cell-to-cell volume transmission mediated by gap junctions between astrocytes bordering CSF compartments and parenchymal astrocytes.5. The width and contents of the interstitial clefts in fetal brain permit cell migration and outgrowth of neurites. The contents of the narrower and different interstitial clefts of mature brain permit solute convection but must be enzymatically degraded in order for cells to migrate through it.     

Fluid flow and convective transport of solutes within the intervertebral disc

Previous experimental and analytical studies of solute transport in the intervertebral disc have demonstrated that for small molecules diffusive transport alone fulfils the nutritional needs of disc cells. It has been often suggested that fluid flow into and within the disc may enhance the transport of larger molecules. The goal of the study was to predict the influence of load-induced interstitial fluid flow on mass transport in the intervertebral disc.An iterative procedure was used to predict the convective transport of physiologically relevant molecules within the disc. An axisymmetric, poroelastic finite-element structural model of the disc was developed. The diurnal loading was divided into discrete time steps. At each time step, the fluid flow within the disc due to compression or swelling was calculated. A sequentially coupled diffusion/convection model was then employed to calculate solute transport, with a constant concentration of solute being provided at the vascularised endplates and outer annulus. Loading was simulated for a complete diurnal cycle, and the relative convective and diffusive transport was compared for solutes with molecular weights ranging from 400 Da to 40 kDa.Consistent with previous studies, fluid flow did not enhance the transport of low-weight solutes. During swelling, interstitial fluid flow increased the unidirectional penetration of large solutes by approximately 100%. Due to the bi-directional temporal nature of disc loading, however, the net effect of convective transport over a full diurnal cycle was more limited (30% increase). Further study is required to determine the significance of large solutes and the timing of their delivery for disc physiology.     

Evidence against cellular internalization in vivo of NMO-IgG, aquaporin-4, and excitatory amino acid transporter 2 in neuromyelitis optica

Autoantibodies against astrocyte water channel aquaporin-4 (AQP4) are thought to be pathogenic in neuromyelitis optica (NMO). Prior work has suggested that a key component of NMO autoantibody (NMO-IgG) pathogenesis is internalization of AQP4 and the associated glutamate transporter EAAT2, leading to glutamate excitotoxicity. Here, we show selective endocytosis of NMO-IgG and AQP4 in transfected cell cultures, but little internalization in brain in vivo. AQP4-dependent endocytosis of NMO-IgG occurred rapidly in various AQP4-transfected cell lines, with efficient transport from early endosomes to lysosomes. Cell surface AQP4 was also reduced following NMO-IgG exposure. However, little or no internalization of NMO-IgG, AQP4, or EAAT2 was found in primary astrocyte cultures, nor was glutamate uptake affected by NMO-IgG exposure. Following injection of NMO-IgG into mouse brain, NMO-IgG binding and AQP4 expression showed a perivascular astrocyte distribution, without detectable cellular internalization over 24 h. We conclude that astrocyte endocytosis of NMO-IgG, AQP4, and EAAT2 is not a significant consequence of AQP4 autoantibody in vivo, challenging generally accepted views about NMO pathogenesis.     

Effect of head-down tilt on brain water distribution

Vascular and tissue fluid dynamics in the microgravity of space environments is commonly simulated by head-down tilt (HDT). Previous reports have indicated that intracranial pressure and extracranial vascular pressures increase during acute HDT and may cause cerebral edema. Tissue water changes within the cranium are detectable by T2 magnetic resonance imaging. We obtained T2 images of sagittal slices from five subjects while they were supine and during -13 degrees HDT using a 1.5-Tesla whole-body magnet. The analysis of difference images demonstrated that HDT leads to a 21% reduction of T2 in the subarachnoid cerebrospinal fluid (CSF) compartment and a 11% reduction in the eyes, which implies a reduction of water content; no increase in T2 was observed in other brain regions that have been associated with cerebral edema. These findings suggest that water leaves the CSF and ocular compartments by exudation as a result of increased transmural pressure causing water to leave the cranium via the spinal CSF compartment or the venous circulation.     

A mixture theory model of fluid and solute transport in the microvasculature of normal and malignant tissues. I. Theory

In order to better understand the mechanisms governing transport of drugs, nanoparticle-based treatments, and therapeutic biomolecules, and the role of the various physiological parameters, a number of mathematical models have previously been proposed. The limitations of the existing transport models indicate the need for a comprehensive model that includes transport in the vessel lumen, the vessel wall, and the interstitial space and considers the effects of the solute concentration on fluid flow. In this study, a general model to describe the transient distribution of fluid and multiple solutes at the microvascular level was developed using mixture theory. The model captures the experimentally observed dependence of the hydraulic permeability coefficient of the capillary wall on the concentration of solutes present in the capillary wall and the surrounding tissue. Additionally, the model demonstrates that transport phenomena across the capillary wall and in the interstitium are related to the solute concentration as well as the hydrostatic pressure. The model is used in a companion paper to examine fluid and solute transport for the simplified case of an axisymmetric geometry with no solid deformation or interconversion of mass.     

Cerebrospinal fluid beta-endorphin in models of hyperalgesia in the rat

Cerebrospinal fluid (CSF) obtained by acute percutaneous puncture of the cisternal membrane of the halothane anesthetized rat has low but measurable concentrations of beta-endorphin-like immunoreactivity (beta-EPir: 32.8 +/- 3.0 pmol/l). Chromatographic separation of beta-EPir showed that authentic beta-endorphin1-31 was the main component of beta-EPir in cisternal CSF. Subcutaneous injection of 5% formalin in the hind paws did not increase beta-EPir in cisternal CSF. Rats with tactile paw hyperalgesia evoked by unilateral ligation of the L5/6 nerve roots 2 weeks earlier had beta-EPir concentrations that did not differ from sham operated or unoperated control animals. In contrast, capsaicin injected in the hindpaws increased the mean beta-EPir concentration compared to saline injections (P = 0.006) 45 min after emerging from anesthesia following injection. These results show that acute activation of C fibers (by capsaicin) will evoke the release of beta-endorphin into the CSF, suggesting activation of the beta-endorphin terminal systems in the brain/midbrain. The failure of formalin injections to release beta-EPir to CSF may be due to specificity of the afferent stimulus evoking beta-EPir release, a lower stimulus intensity, and/or the duration of the stimulus generated by formalin. The normal concentrations of beta-EPir found in the hyperalgesic state following nerve injury suggest that the supraspinal beta-endorphin system does not display tonic changes under such conditions.     

Cellular uptake of intracerebroventricularly administered biotin- or digoxigenin-Labeled antisense oligodeoxynucleotides in the rat

Summary 1. Antisense oligodeoxynucleotides (ODNs) internally labeled with biotin or digoxigenin were injected into the lateral ventricle of rats and the distribution of the labeled ODNs was examined at several timepoints following the intracerebroventricular (icv) injections. The stability of these injected antisense ODNs, which had no backbone modifications, was also studied by performing recovery experiments.2. The most intense labeling was observed near the injection site, in periventricular areas, and in perivascular regions. Many of the labeled cells appeared to be neurons, and both the cytoplasm and the nuclei were stained. The labeled cells were detected 15 min after icv injection, demonstrating that the antisense ODNs were taken up rapidly by cells in the parenchyma. The digoxigeninated antisense ODNs were presented in both the cytoplasmic and the nuclear fractions of rat brain extracts, however, the levels appeared to be much lower in the nuclear fractions.3. Antisense ODNs injected into the lateral ventricle seemed to follow the bulk flow of cerebrospinal fluid (CSF), i.e., from the injection site in the lateral ventricle, through the ventricular system, to the subarachnoid spaces and the perivascular spaces. From the ventricular and perivascular spaces, the antisense ODNs diffused into the extracellular space and were taken up by cells. The full-length digoxigeninated antisense ODNs were detectable within cells after only 15 min, indicating their rapid uptake. In addition, the antisense ODNs appeared to be relatively stable in the brain since the full-length digoxigeninated ODNs were still detectable after 4 hr.     

Carrier-Mediated Transport of Chloride Across the Blood-Brain Barrier

36Cl concentrations in each of eight brain regions and in cisternal cerebrospinal fluid (CSF) were determined 30 min after the intravenous injection of 36Cl in dialyzed-nephrectomized rats with plasma Cl concentrations between 14 and 120 mumol X ml-1. CSF 36Cl exceeded 36Cl concentrations in brain extracellular fluid. The calculated blood-to-brain transfer constants for Cl, kCl, ranged from 1.8 X 10(-5) S-1 at the parietal cortex to 3.8 X 10(-5) S-1 at the thalamus-hypothalamus. kCl fell by 42-62% when mean plasma [Cl] was elevated from 16 to 114 mumol X ml-1. Brain uptake of [14C]mannitol or of 22Na was independent of plasma [Cl], but 22Na influx into CSF fell when plasma [Cl] was reduced. Cl flux into brain and CSF could be represented by Michaelis-Menten saturation kinetics, where, for the parietal cortex, Km = 43 mumol X ml-1 and Vmax = 2.5 X 10(-3) mumol X S-1 X g-1, and for CSF Km = 68 mumol X ml-1. At least 80% of 36Cl influx into the parietal cortex was calculated to occur at the cerebrovascular endothelium, whereas the remainder was derived from tracer that first entered CSF. The CSF contribution was greater at brain regions adjacent to cerebral ventricles. The results show that Cl transport at the cerebrovascular endothelium as well as at the choroid plexus epithelium is a saturable concentration-dependent process, and that the CSF is a significant intermediate pathway for Cl passage from blood to brain.