Response to Commentary on "Structural characterization of SLYM - a 4th meningeal membrane" by Julie Siegenthaler and Christer Betsholtz.

Histological studies have for decades documented that each of the classical meningeal membranes contains multiple fibroblast layers with distinct cellular morphology. Particularly, the sublayers of the arachnoid membranes have received attention due to their anatomical complexity. Early studies found that tracers injected into the cerebrospinal fluid (CSF) do not distribute freely but are restricted by the innermost sublayer of the arachnoid membrane. The existence of restrictions on CSF movement and the subdivision of the subarachnoid space into several distinct compartments have recently been confirmed by in vivo 2-photon studies of rodents, as well as macroscopic imaging of pigs and magnetic resonance imaging of human brain. Based on in vivo imaging and immunophenotyping characterization, we identified the structural basis for this compartmentalization of the subarachnoid space, which we term 'Subarachnoid lymphatic-like membrane', SLYM. The SLYM layer engages the subarachnoid vasculature as it approaches the brain parenchyma, demarcating a roof over pial perivascular spaces. Functionally, the separation of pial periarterial and perivenous spaces in the larger subarachnoid space is critical for the maintenance of unidirectional glymphatic clearance. In light of its close apposition to the pial surface and to the brain perivascular fluid exit points, the SLYM also provides a primary locus for immune surveillance of the brain. Yet, the introduction of SLYM, in terms of its anatomic distinction and hence functional specialization, has met resistance. Its critics assert that SLYM has been described in the literature by other terms, including the inner arachnoid membrane, the interlaminate membrane, the outer pial layer, the intermediate lamella, the pial membrane, the reticular layer of the arachnoid membrane or, more recently, BFB2-3. We argue that our conception of SLYM as an anatomically and functionally distinct construct is both necessary and warranted since its functional roles are wholly distinct from those of the overlying arachnoid barrier layer. Our terminology also lends clarity to a complex anatomy that has hitherto been ill-described. In that regard, we also note the lack of specificity of DPP4, which has recently been introduced as a 'selected defining marker' of the arachnoid barrier layer. We note that DPP4 labels fibroblasts in all meningeal membranes as well as in the trabecula arachnoides and the vascular adventitial layers, thus obviating its utility in meningeal characterization. Instead, we report a set of glymphatic-associated proteins that serve to accurately specify SLYM and distinguish it from its adjacent yet functionally distinct membranes.

Restoration of cervical lymphatic vessel function in aging rescues cerebrospinal fluid drainage.

Cervical lymphatic vessels (cLVs) have been shown to drain solutes and cerebrospinal fluid (CSF) from the brain. However, their hydrodynamical properties have never been evaluated in vivo. Here, we developed two-photon optical imaging with particle tracking in vivo of CSF tracers (2P-OPTIC) in superficial and deep cLVs of mice, characterizing their flow and showing that the major driver is intrinsic pumping by contraction of the lymphatic vessel wall. Moreover, contraction frequency and flow velocity were reduced in aged mice, which coincided with a reduction in smooth muscle actin expression. Slowed flow in aged mice was rescued using topical application of prostaglandin F2α, a prostanoid that increases smooth muscle contractility, which restored lymphatic function in aged mice and enhanced central nervous system clearance. We show that cLVs are important regulators of CSF drainage and that restoring their function is an effective therapy for improving clearance in aging.

A systematic review and meta-analysis of English language online patient education materials in breast cancer: Is readability the only story?

BACKGROUND: Online patient education materials (OPEMs) are an increasingly popular resource for women seeking information about breast cancer. The AMA recommends written patient material to be at or below a 6th grade level to meet the general public's health literacy. Metrics such as quality, understandability, and actionability also heavily influence the usability of health information, and thus should be evaluated alongside readability. PURPOSE: A systematic review and meta-analysis was conducted to determine: 1) Average readability scores and reporting methodologies of breast cancer readability studies; and 2) Inclusion frequency of additional health literacy-associated metrics. MATERIALS AND METHODS: A registered systematic review and meta-analysis was conducted in Ovid MEDLINE, Web of Science, Embase.com, CENTRAL via Ovid, and ClinicalTrials.gov in June 2022 in adherence with the PRISMA 2020 statement. Eligible studies performed readability analyses on English-language breast cancer-related OPEMs. Study characteristics, readability data, and reporting of non-readability health literacy metrics were extracted. Meta-analysis estimates were derived from generalized linear mixed modeling. RESULTS: The meta-analysis included 30 studies yielding 4462 OPEMs. Overall, average readability was 11.81 (95% CI [11.14, 12.49]), with a significant difference (p < 0.001) when grouped by OPEM categories. Commercial organizations had the highest average readability at 12.2 [11.3,13.0]; non-profit organizations had one of the lowest at 11.3 [10.6,12.0]. Readability also varied by index, with New Fog, Lexile, and FORCAST having the lowest average scores (9.4 [8.6, 10.3], 10.4 [10.0, 10.8], and 10.7 [10.2, 11.1], respectively). Only 57% of studies calculated average readability with more than two indices. Only 60% of studies assessed other OPEM metrics associated with health literacy. CONCLUSION: Average readability of breast cancer OPEMs is nearly double the AMA's recommended 6th grade level. Readability and other health literacy-associated metrics are inconsistently reported in the current literature. Standardization of future readability studies, with a focus on holistic evaluation of patient materials, may aid shared decision-making and be critical to increased screening rates and breast cancer awareness.

Transient but not chronic hyperglycemia accelerates ocular glymphatic transport.

Glymphatic transport is vital for the physiological homeostasis of the retina and optic nerve. Pathological alterations of ocular glymphatic fluid transport and enlarged perivascular spaces have been described in glaucomatous mice. It remains to be established how diabetic retinopathy, which impairs vision in about 50% of diabetes patients, impacts ocular glymphatic fluid transport. Here, we examined ocular glymphatic transport in chronic hyperglycemic diabetic mice as well as in healthy mice experiencing a daily transient increase in blood glucose. Mice suffering from severe diabetes for two and four months, induced by streptozotocin, exhibited no alterations in ocular glymphatic fluid transport in the optic nerve compared to age-matched, non-diabetic controls. In contrast, transient increases in blood glucose induced by repeated daily glucose injections in healthy, awake, non-diabetic mice accelerated antero- and retrograde ocular glymphatic transport. Structural analysis showed enlarged perivascular spaces in the optic nerves of glucose-treated mice, which were absent in diabetic mice. Thus, transient repeated hyperglycemic events, but not constant hyperglycemia, ultimately enlarge perivascular spaces in the murine optic nerve. These findings indicate that fluid transport in the mouse eye is vulnerable to fluctuating glycemic levels rather than constant hyperglycemia, suggesting that poor glycemic control drives glymphatic malfunction and perivascular enlargement in the optic nerve.

Structural characterization of SLYM-a 4th meningeal membrane.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4th meningeal membrane, Subarachnoid Lymphatic-like Membrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pia-adjacent vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that, in spinal cord and parts of dorsal cortex, SLYM fused with the arachnoid barrier layer, while in the basal brain stem typically formed a 1-3 cell layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpreting the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of these fragile membranes in postmortem preparations.

Reemplazo percutáneo de válvulas cardíacas derechas con técnica "válvula en válvula" en cardiopatías congénitas: experiencia inicial en Argentina

INTRODUCCIÓN: El reemplazo quirúrgico con colocación de bioprótesis es una estrategia utilizada frecuentemente para tratar valvulopatías en cardiopatías congénitas. OBJETIVO: Presentar la experiencia inicial en Argentina con fractura/remodelación del anillo valvular en bioprótesis y posterior reemplazo percutáneo "válvula en válvula" en pacientes con cardiopatías congénitas. MATERIAL y métodos: Estudio descriptivo y observacional. Se incluyeron todos los pacientes tratados con reemplazo percutáneo debido a disfunción de prótesis valvular biológica. RESULTADOS: Desde agosto de 2021 hasta mayo de 2023, 5 pacientes (3 de sexo femenino) con disfunción de bioprótesis derechas recibieron tratamiento percutáneo como alternativa a la realización de un nuevo reemplazo quirúrgico. La edad media fue 21,2 ± 9,2 años, el peso medio fue 56,2 ± 22,2 kg. Fueron implantadas cinco válvulas balón expandibles: 3 en posición pulmonar y 2 en posición tricúspide y en 4 casos, con fractura previa del anillo valvular. En todos los pacientes se restauró la función valvular, sin complicaciones. En el seguimiento, en un sólo paciente se objetivó insuficiencia valvular moderada derecha CONCLUSIÓN: El reemplazo percutáneo "válvula en válvula" en bioprótesis disfuncionantes derechas es una estrategia atractiva y segura en casos seleccionados, que restaura la competencia y elimina las obstrucciones. Esta estrategia es una opción razonable como alternativa a la realización de un nuevo recambio valvular quirúrgico.

Structural characterization of SLYM - a 4th meningeal membrane.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4th meningeal membrane, Subarachnoid Lymphatic-like Membrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pia-adjacent vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that, in spinal cord and parts of dorsal cortex, SLYM fused with the arachnoid barrier layer, while in the basal brain stem typically formed a 1-3 cell layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpreting the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of these fragile membranes in postmortem preparations.

Structural characterization of SLYM - a 4 th meningeal membrane.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4 th meningeal membrane, S ubarachnoid Ly mphatic-like M embrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pia-adjacent vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that, in spinal cord and parts of dorsal cortex, SLYM fused with the arachnoid barrier layer, while in the basal brain stem typically formed a 1-3 cell layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpreting the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of these fragile membranes in postmortem preparations.

Sizes and shapes of perivascular spaces surrounding murine pial arteries.

BACKGROUND: Flow of cerebrospinal fluid (CSF) through brain perivascular spaces (PVSs) is essential for the clearance of interstitial metabolic waste products whose accumulation and aggregation is a key mechanism of pathogenesis in many diseases. The PVS geometry has important implications for CSF flow as it affects CSF and solute transport rates. Thus, the size and shape of the perivascular spaces are essential parameters for models of CSF transport in the brain and require accurate quantification. METHODS: We segmented two-photon images of pial (surface) PVSs and the adjacent arteries and characterized their sizes and shapes of cross sections from 14 PVS segments in 9 mice. Based on the analysis, we propose an idealized model that approximates the cross-sectional size and shape of pial PVSs, closely matching their area ratios and hydraulic resistances. RESULTS: The ratio of PVS-to-vessel area varies widely across the cross sections analyzed. The hydraulic resistance per unit length of the PVS scales with the PVS cross-sectional area, and we found a power-law fit that predicts resistance as a function of the area. Three idealized geometric models were compared to PVSs imaged in vivo, and their accuracy in reproducing hydraulic resistances and PVS-to-vessel area ratios were evaluated. The area ratio was obtained across different cross sections, and we found that the distribution peaks for the original PVS and its closest idealized fit (polynomial fit) were 1.12 and 1.21, respectively. The peak of the hydraulic resistance distribution is [Formula: see text] Pa  s/m[Formula: see text] and [Formula: see text] Pa s/m[Formula: see text] for the segmentation and its closest idealized fit, respectively. CONCLUSIONS: PVS hydraulic resistance can be reasonably predicted as a function of the PVS area. The proposed polynomial-based fit most closely captures the shape of the PVS with respect to area ratio and hydraulic resistance. Idealized PVS shapes are convenient for modeling, which can be used to better understand how anatomical variations affect clearance and drug transport.

Astrocyte endfeet may theoretically act as valves to convert pressure oscillations to glymphatic flow.

The glymphatic system of cerebrospinal fluid transport through the perivascular spaces of the brain has been implicated in metabolic waste clearance, neurodegenerative diseases and in acute neurological disorders such as stroke and cardiac arrest. In other biological low-pressure fluid pathways such as in veins and the peripheral lymphatic system, valves play an important role in ensuring the flow direction. Though fluid pressure is low in the glymphatic system and directed bulk flow has been measured in pial and penetrating perivascular spaces, no valves have yet been identified. Valves, which asymmetrically favour forward flow to backward flow, would imply that the considerable oscillations in blood and ventricle volumes seen in magnetic resonance imaging could cause directed bulk flow. Here, we propose that astrocyte endfeet may act as such valves using a simple elastic mechanism. We combine a recent fluid mechanical model of viscous flow between elastic plates with recent measurements of in vivo elasticity of the brain to predict order of magnitude flow-characteristics of the valve. The modelled endfeet are effective at allowing forward while preventing backward flow.

Mammography Screening Outreach Through Non-Primary Care-Based Services.

OBJECTIVE: To estimate the proportion of patients visiting urgent care centers or emergency departments or being hospitalized who were not up to date with recommended mammography screening to assess the potential impact of non-primary care-based cancer screening interventions. METHODS: Adult participants from the 2019 National Health Interview Survey were included. Among participants not up to date with breast cancer screening guidelines based on ACR recommendations, the proportion of patients reporting an urgent care, emergency department visit, or hospitalization within the last year was estimated accounting for complex survey sampling design features. Multiple variable logistic regression analyses were then conducted to evaluate the association between sociodemographic characteristics and mammography screening adherence. RESULTS: The study included 9,139 women between the ages of 40 and 74 years without history of breast cancer. Of these respondents, 44.9% did not report mammography screening within the last year. Among participants who did not report mammography screening, 29.2% reported visiting an urgent care center, 21.8% reported visiting an emergency room, and 9.6% reported being hospitalized within the last year. The majority of patients receiving non-primary care-based services, who were not up to date with mammography screening, were from historically underserved groups including Black and Hispanic patients. CONCLUSION: Nearly 10% to 30% of participants who have not obtained recommended breast cancer screening have visited non-primary care-based services including urgent care centers or emergency rooms or have been hospitalized within the last year.

Astrocytic chloride is brain state dependent and modulates inhibitory neurotransmission in mice.

Information transfer within neuronal circuits depends on the balance and recurrent activity of excitatory and inhibitory neurotransmission. Chloride (Cl-) is the major central nervous system (CNS) anion mediating inhibitory neurotransmission. Astrocytes are key homoeostatic glial cells populating the CNS, although the role of these cells in regulating excitatory-inhibitory balance remains unexplored. Here we show that astrocytes act as a dynamic Cl- reservoir regulating Cl- homoeostasis in the CNS. We found that intracellular chloride concentration ([Cl-]i) in astrocytes is high and stable during sleep. In awake mice astrocytic [Cl-]i is lower and exhibits large fluctuation in response to both sensory input and motor activity. Optogenetic manipulation of astrocytic [Cl-]i directly modulates neuronal activity during locomotion or whisker stimulation. Astrocytes thus serve as a dynamic source of extracellular Cl- available for GABAergic transmission in awake mice, which represents a mechanism for modulation of the inhibitory tone during sustained neuronal activity.

Artificial intelligence velocimetry reveals in vivo flow rates, pressure gradients, and shear stresses in murine perivascular flows.

Quantifying the flow of cerebrospinal fluid (CSF) is crucial for understanding brain waste clearance and nutrient delivery, as well as edema in pathological conditions such as stroke. However, existing in vivo techniques are limited to sparse velocity measurements in pial perivascular spaces (PVSs) or low-resolution measurements from brain-wide imaging. Additionally, volume flow rate, pressure, and shear stress variation in PVSs are essentially impossible to measure in vivo. Here, we show that artificial intelligence velocimetry (AIV) can integrate sparse velocity measurements with physics-informed neural networks to quantify CSF flow in PVSs. With AIV, we infer three-dimensional (3D), high-resolution velocity, pressure, and shear stress. Validation comes from training with 70% of PTV measurements and demonstrating close agreement with the remaining 30%. A sensitivity analysis on the AIV inputs shows that the uncertainty in AIV inferred quantities due to uncertainties in the PVS boundary locations inherent to in vivo imaging is less than 30%, and the uncertainty from the neural net initialization is less than 1%. In PVSs of N = 4 wild-type mice we find mean flow speed 16.33 ± 11.09 µm/s, volume flow rate 2.22 ± 1.983 × 103 µm3/s, axial pressure gradient ( - 2.75 ± 2.01)×10-4 Pa/µm (-2.07 ± 1.51 mmHg/m), and wall shear stress (3.00 ± 1.45)×10-3 Pa (all mean ± SE). Pressure gradients, flow rates, and resistances agree with prior predictions. AIV infers in vivo PVS flows in remarkable detail, which will improve fluid dynamic models and potentially clarify how CSF flow changes with aging, Alzheimer's disease, and small vessel disease.

Sizes and Shapes of Perivascular Spaces Surrounding Murine Pial Arteries.

Background: Flow of cerebrospinal fluid (CSF) through brain perivascular spaces (PVSs) is essential for the clearance of interstitial metabolic waste products whose accumulation and aggregation is a key mechanism of pathogenesis in many diseases. The PVS geometry has important implications for CSF flow as it affects CSF and solute transport rates. Thus, the size and shape of the perivascular spaces are essential parameters for models of CSF transport in the brain and require accurate quantification. Methods: We segmented two-photon images of pial (surface) PVSs and the adjacent arteries and characterized their sizes and shapes of thousands of cross sections from 14 PVS segments in 9 mice. Based on the analysis, we propose an idealized model that approximates the cross-sectional size and shape of pial PVSs, closely matching their area ratios and hydraulic resistances. Results: PVS size only approximately scales with vessel size, and the ratio of PVS-to-vessel area varies widely across the thousands of cross sections analyzed. The hydraulic resistance per unit length of the PVS scales with the PVS cross-sectional area, and we found a power-law fit that predicts resistance as a function of the area. Three idealized geometric models were compared to PVSs imaged in vivo, and their accuracy in reproducing hydraulic resistances and PVS-to-vessel area ratios were evaluated. The area ratio was obtained across thousands of different cross sections, and we found that the distribution peaks for the original PVS and its closest idealized fit (polynomial fit) were 1.12 and 1.21, respectively. The peak of the hydraulic resistance distribution is 1.73 x 10 15 Pa-s/m 5 and 1.44 x 10 15 Pa-s/m 5 for the segmentation and its closest idealized fit, respectively. Conclusions: Brief summary and potential implicationsPVS hydraulic resistance can be reasonably predicted as a function of the PVS area. The proposed polynomial-based fit most closely captures the shape of the PVS with respect to area ratio and hydraulic resistance. Idealized PVS shapes are convenient for modeling, which can be used to better understand how anatomical variations affect clearance and drug delivery transport.

Percutaneous Closure of Ruptured Sinus of Valsalva Aneurysm in a Child Using a Patent Ductus Arteriosus Device.

Ruptured sinus of Valsalva aneurysm is rare, although if left untreated, potentially fatal disease. Surgical approach has been the main treatment in most series; nevertheless, percutaneous closure has been described in selected cases. We report a 5-year-old boy presenting with rapid clinical deterioration who underwent percutaneous closure using a patent ductus arteriosus device, with the resolution of symptoms. Descriptions of this technique being utilized in children are infrequent in the literature.

Tetralogy of Fallot Repair After Neonatal Right Ventricular Outflow Tract Stenting: Initial Multicenter Experience in Argentina.

Initial management of patients with tetralogy of Fallot, unfavorable anatomy, and reduced pulmonary blood flow is controversial and continues to be a clinical challenge. Pulmonary to systemic shunt anastomosis or primary correction in neonates and small infants is associated with higher morbimortality and increased number of reoperations. Initial right ventricle outflow tract stenting palliation has emerged as an attractive alternative. We report our experience in 14 patients operated on with tetralogy of Fallot and previous right ventricle outflow tract stenting from March 2018 to June 2022. All stented patients had pulmonary annulus and main pulmonary artery Z score ≤ -2.5. Surgical outcomes, complications, and mortality at 30 days were evaluated. Patient's age and weight at surgery were 5.9 months (2-17) and 6.1 kg (3.9-8.9), respectively. Stents were completely removed in 57.1% of patients. A transannular patch was placed in 10 patients, 3 patients required a right ventricle to pulmonary artery conduit due to coronary anomalies and in 1 patient, the pulmonary valve was preserved. Length of stay and ventilation time were 13.6 days (5-27) and 44.8 h (6-44), respectively. Mean time for right ventricle outflow tract stent implantation to surgical correction was 4 months (2-16). There was no mortality, and mean follow-up time of this cohort was 23.1 month (1-41). Surgical correction of severe tetralogy of Fallot after right ventricle outflow tract stenting is an effective alternative achievable without an increase in morbidity and mortality. Difficulty in stent extraction is related to the time since implantation. More number of patients and longer follow-up time are needed to confirm these initial results.

Prevalence of Modifiable Breast Cancer Risk Factors and Potential Opportunities for Primary Prevention Among Women Engaged in Screening Mammography: National Health Interview Survey Results.

OBJECTIVE: To determine the prevalence of modifiable breast cancer risk factors among women engaged in screening mammography using nationally representative cross-sectional survey data and to inform potential opportunities for breast facilities to contribute to primary prevention. METHODS: 2018 National Health Interview Survey respondents who were women ages 40-74 years without history of breast cancer were included and then categorized based on whether they reported screening mammography within the prior two years. Proportions of these women reporting evidence-based modifiable breast cancer risk factors, including elevated body mass index (BMI), lack of physical activity, or moderate or heavy alcohol consumption were calculated and stratified by demographics. Multivariable logistic regression was used to estimate the association between these risk factors and sociodemographic characteristics. RESULTS: Among 4989 women meeting inclusion criteria and reporting screening mammography, 79% reported at least one modifiable risk factor. Elevated BMI was the most reported risk factor (67%), followed by lack of physical activity (24%) and alcohol consumption (16%). The majority of each race/ethnicity category reported at least one modifiable risk factor, with the highest proportion reported by Black respondents (90%). Asian, college educated, and higher-income participants were less likely to have at least one modifiable risk factor. CONCLUSION: Modifiable breast cancer risk factors are prevalent among women engaged in screening mammography. This provides potential opportunities for breast imaging facilities to contribute to the primary prevention of breast cancer by providing resources for lifestyle modification at the time of screening mammography.

Measurements of cerebrospinal fluid production: a review of the limitations and advantages of current methodologies.

Cerebrospinal fluid (CSF) is an essential and critical component of the central nervous system (CNS). According to the concept of the "third circulation" originally proposed by Cushing, CSF is mainly produced by the choroid plexus and subsequently leaves the cerebral ventricles via the foramen of Magendie and Luschka. CSF then fills the subarachnoid space from whence it disperses to all parts of the CNS, including the forebrain and spinal cord. CSF provides buoyancy to the submerged brain, thus protecting it against mechanical injury. CSF is also transported via the glymphatic pathway to reach deep interstitial brain regions along perivascular channels; this CSF clearance pathway promotes transport of energy metabolites and signaling molecules, and the clearance of metabolic waste. In particular, CSF is now intensively studied as a carrier for the removal of proteins implicated in neurodegeneration, such as amyloid-ß and tau. Despite this key function of CSF, there is little information about its production rate, the factors controlling CSF production, and the impact of diseases on CSF flux. Therefore, we consider it to be a matter of paramount importance to quantify better the rate of CSF production, thereby obtaining a better understanding of CSF dynamics. To this end, we now review the existing methods developed to measure CSF production, including invasive, noninvasive, direct, and indirect methods, and MRI-based techniques. Depending on the methodology, estimates of CSF production rates in a given species can extend over a ten-fold range. Throughout this review, we interrogate the technical details of CSF measurement methods and discuss the consequences of minor experimental modifications on estimates of production rate. Our aim is to highlight the gaps in our knowledge and inspire the development of more accurate, reproducible, and less invasive techniques for quantitation of CSF production.

A real-time in vivo clearance assay for quantification of glymphatic efflux.

Glymphatic fluid transport eliminates metabolic waste from the brain including amyloid-ß, yet the methodology for studying efflux remains rudimentary. Here, we develop a method to evaluate glymphatic real-time clearance. Efflux of Direct Blue 53 (DB53, also T-1824 or Evans Blue) injected into the striatum is quantified by imaging the DB53 signal in the vascular compartment, where it is retained due to its high affinity to albumin. The DB53 signal is detectable as early as 15 min after injection and the efflux kinetics are sharply reduced in mice lacking the water channel aquaporin 4 (AQP4). Pharmacokinetic modeling reveal that DB53 efflux is consistent with the existence of two efflux paths, one with fast kinetics (T1/2 = 50 min) and another with slow kinetics (T1/2 = 240 min), in wild-type mice. This in vivo methodology will aid in defining the physiological variables that drive efflux, as well as the impact of brain states or disorders on clearance kinetics.