Interstitial Fluid Shear Stress Induces the Synthetic Phenotype Switching of VSMCs to Release Pro-calcified Extracellular Vesicles via EGFR-MAPK-KLF5 Pathway.

Phenotypic switching (from contractile to synthetic) of vascular smooth muscle cells (VSMCs) is essential in the progression of atherosclerosis. The damaged endothelium in the atherosclerotic artery exposes VSMCs to increased interstitial fluid shear stress (IFSS). However, the precise mechanisms by which increased IFSS influences VSMCs phenotypic switching are unrevealed. Here, we employed advanced numerical simulations to calculate IFSS values accurately based on parameters acquired from patient samples. We then carefully investigated the phenotypic switching and extracellular vesicles (EVs) secretion of VSMCs under various IFSS conditions. By employing a comprehensive set of approaches, we found that VSMCs exhibited synthetic phenotype upon atherosclerotic IFSS. This synthetic phenotype is the upstream regulator for the enhanced secretion of pro-calcified EVs. Mechanistically, as a mechanotransducer, the epidermal growth factor receptor (EGFR) initiates the flow-based mechanical cues to MAPK signaling pathway, facilitating the nuclear accumulation of the transcription factor krüppel-like factor 5 (KLF5). Furthermore, pharmacological inhibiting either EGFR or MAPK signaling pathway blocks the nuclear accumulation of KLF5 and finally results in the maintenance of contractile VSMCs even under increased IFSS stimulation. Collectively, targeting this signaling pathway holds potential as a novel therapeutic strategy to inhibit VSMCs phenotypic switching and mitigate the progression of atherosclerosis.

Curvature-mediated rapid extravasation and penetration of nanoparticles against interstitial fluid pressure for improved drug delivery.

Elevated interstitial fluid pressure (IFP) within pathological tissues (e.g., tumors, obstructed kidneys, and cirrhotic livers) creates a significant hindrance to the transport of nanomedicine, ultimately impairing the therapeutic efficiency. Among these tissues, solid tumors present the most challenging scenario. While several strategies through reducing tumor IFP have been devised to enhance nanoparticle delivery, few approaches focus on modulating the intrinsic properties of nanoparticles to effectively counteract IFP during extravasation and penetration, which are precisely the stages obstructed by elevated IFP. Herein, we propose an innovative solution by engineering nanoparticles with a fusiform shape of high curvature, enabling efficient surmounting of IFP barriers during extravasation and penetration within tumor tissues. Through experimental and theoretical analyses, we demonstrate that the elongated nanoparticles with the highest mean curvature outperform spherical and rod-shaped counterparts against elevated IFP, leading to superior intratumoral accumulation and antitumor efficacy. Super-resolution microscopy and molecular dynamics simulations uncover the underlying mechanisms in which the high curvature contributes to diminished drag force in surmounting high-pressure differentials during extravasation. Simultaneously, the facilitated rotational movement augments the hopping frequency during penetration. This study effectively addresses the limitations posed by high-pressure impediments, uncovers the mutual interactions between the physical properties of NPs and their environment, and presents a promising avenue for advancing cancer treatment through nanomedicine.

Flow in temporally and spatially varying porous media: a model for transport of interstitial fluid in the brain.

Flow in a porous medium can be driven by the deformations of the boundaries of the porous domain. Such boundary deformations locally change the volume fraction accessible by the fluid, creating non-uniform porosity and permeability throughout the medium. In this work, we construct a deformation-driven porous medium transport model with spatially and temporally varying porosity and permeability that are dependent on the boundary deformations imposed on the medium. We use this model to study the transport of interstitial fluid along the basement membranes in the arterial walls of the brain. The basement membrane is modeled as a deforming annular porous channel with the compressible pore space filled with an incompressible, Newtonian fluid. The role of a forward propagating peristaltic heart pulse wave and a reverse smooth muscle contraction wave on the flow within the basement membranes is investigated. Our results identify combinations of wave amplitudes that can induce either forward or reverse transport along these transport pathways in the brain. The magnitude and direction of fluid transport predicted by our model can help in understanding the clearance of fluids and solutes along the Intramural Periarterial Drainage route and the pathology of cerebral amyloid angiopathy.

Chloride ions in health and disease.

Chloride is a key anion involved in cellular physiology by regulating its homeostasis and rheostatic processes. Changes in cellular Cl- concentration result in differential regulation of cellular functions such as transcription and translation, post-translation modifications, cell cycle and proliferation, cell volume, and pH levels. In intracellular compartments, Cl- modulates the function of lysosomes, mitochondria, endosomes, phagosomes, the nucleus, and the endoplasmic reticulum. In extracellular fluid (ECF), Cl- is present in blood/plasma and interstitial fluid compartments. A reduction in Cl- levels in ECF can result in cell volume contraction. Cl- is the key physiological anion and is a principal compensatory ion for the movement of the major cations such as Na+, K+, and Ca2+. Over the past 25 years, we have increased our understanding of cellular signaling mediated by Cl-, which has helped in understanding the molecular and metabolic changes observed in pathologies with altered Cl- levels. Here, we review the concentration of Cl- in various organs and cellular compartments, ion channels responsible for its transportation, and recent information on its physiological roles.

A Method to Collect Cerebrospinal Fluid from Mouse Cisterna Magna to Determine Extracellular Tau Levels.

Despite being a cytoplasmic protein abundant in neurons, tau is detectable in various extracellular fluids. In addition to being passively released from dying/degenerating neurons, tau is also actively released from living neurons in a neuronal activity-dependent mechanism. In vivo, tau released from neurons first appears in brain interstitial fluid (ISF) and subsequently drains into cerebrospinal fluid (CSF) by glymphatic system. Changes in CSF tau levels alter during the course of AD pathogenesis and are considered to predict the disease-progression of AD. A method to collect CSF from various mouse models of AD will serve as a valuable tool to investigate the dynamics of physiological/pathological tau released from neurons. In this chapter, we describe and characterize a method that reliably collects a relatively large volume of CSF from anesthetized mice.

Detection of Glymphatic Outflow of Tau from Brain to Cerebrospinal Fluid in Mice.

Glymphatic system denotes a brain-wide pathway that eliminates extracellular solutes from brain. It is driven by the flow of brain interstitial fluid (ISF) and cerebrospinal fluid (CSF) via perivascular spaces. Glymphatic convective flow is driven by cerebral arterial pulsation, which is facilitated by a water channel, aquaporin-4 (AQP4) expressed in astrocytic end-foot processes. Since its discovery, the glymphatic system receives a considerable scientific attention due to its pivotal role in clearing metabolic waste as well as neurotoxic substances such as amyloid b peptide. Tau is a microtubule binding protein, however it is also physiologically released into extracellular fluids. The presence of tau in the blood stream indicates that it is eventually cleared from the brain to the periphery, however, the detailed mechanisms that eliminate extracellular tau from the central nervous system remained to be elucidated. Recently, we and others have reported that extracellular tau is eliminated from the brain to CSF by an AQP4 dependent mechanism, suggesting the involvement of the glymphatic system. In this chapter, we describe the detailed protocol of how we can assess glymphatic outflow of tau protein from brain to CSF in mice.

On-Site Melanoma Diagnosis Utilizing a Swellable Microneedle-Assisted Skin Interstitial Fluid Sampling and a Microfluidic Particle Dam for Visual Quantification of S100A1.

Malignant melanoma (MM) is the most aggressive form of skin cancer. The delay in treatment will induce metastasis, resulting in a poor prognosis and even death. Here, a two-step strategy for on-site diagnosis of MM is developed based on the extraction and direct visual quantification of S100A1, a biomarker for melanoma. First, a swellable microneedle is utilized to extract S100A1 in skin interstitial fluid (ISF) with minimal invasion. After elution, antibody-conjugated magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) are introduced. A high expression level of S100A1 gives rise to a robust binding between MMPs and PMPs and reduces the number of free PMPs. By loading the reacted solution into the device with a microfluidic particle dam, the quantity of free PMPs after magnetic separation is displayed with their accumulation length inversely proportional to S100A1 levels. A limit of detection of 18.7 ng mL-1 for S100A1 is achieved. The animal experiment indicates that ISF-based S100A1 quantification using the proposed strategy exhibits a significantly higher sensitivity compared with conventional serum-based detection. In addition, the result is highly comparable with the gold standard enzyme-linked immunosorbent assay based on Lin's concordance correlation coefficient, suggesting the high practicality for routine monitoring of melanoma.

Multisensory gamma stimulation promotes glymphatic clearance of amyloid.

The glymphatic movement of fluid through the brain removes metabolic waste1-4. Noninvasive 40 Hz stimulation promotes 40 Hz neural activity in multiple brain regions and attenuates pathology in mouse models of Alzheimer's disease5-8. Here we show that multisensory gamma stimulation promotes the influx of cerebrospinal fluid and the efflux of interstitial fluid in the cortex of the 5XFAD mouse model of Alzheimer's disease. Influx of cerebrospinal fluid was associated with increased aquaporin-4 polarization along astrocytic endfeet and dilated meningeal lymphatic vessels. Inhibiting glymphatic clearance abolished the removal of amyloid by multisensory 40 Hz stimulation. Using chemogenetic manipulation and a genetically encoded sensor for neuropeptide signalling, we found that vasoactive intestinal peptide interneurons facilitate glymphatic clearance by regulating arterial pulsatility. Our findings establish novel mechanisms that recruit the glymphatic system to remove brain amyloid.

Neuronal dynamics direct cerebrospinal fluid perfusion and brain clearance.

The accumulation of metabolic waste is a leading cause of numerous neurological disorders, yet we still have only limited knowledge of how the brain performs self-cleansing. Here we demonstrate that neural networks synchronize individual action potentials to create large-amplitude, rhythmic and self-perpetuating ionic waves in the interstitial fluid of the brain. These waves are a plausible mechanism to explain the correlated potentiation of the glymphatic flow1,2 through the brain parenchyma. Chemogenetic flattening of these high-energy ionic waves largely impeded cerebrospinal fluid infiltration into and clearance of molecules from the brain parenchyma. Notably, synthesized waves generated through transcranial optogenetic stimulation substantially potentiated cerebrospinal fluid-to-interstitial fluid perfusion. Our study demonstrates that neurons serve as master organizers for brain clearance. This fundamental principle introduces a new theoretical framework for the functioning of macroscopic brain waves.

Design of in vitro biomimetic experimental system and simulation analysis for transvascular transport of nano-preparation.

Recently, the enhanced penetration and retention (EPR) effect of nano-preparations has been questioned. Whether the vascular endothelial cell gap (VECG) is the main transport pathway of nano-preparations has become a hot issue at present. Therefore, we propose an in vitro biomimetic experimental system that demonstrates the transvascular transport of nano-preparation. Based on the tumor growth process, the experimental system was used to simulate the change process of abnormal factors (vascular endothelial cell gap and interstitial fluid pressure (IFP)) in the tumor microenvironment. The influence of change in the abnormal factors on the enhanced penetration and retention effect of nano-preparation was explored, and simulation verification was performed. The results show that when the interstitial fluid pressure is close to the vascular fluid pressure (VFP), the transport of nano-preparation is obstructed, resulting in the disappearance of enhanced penetration and retention effect of the nano-preparation. This indicates that the pressure gradient between vascular fluid pressure and interstitial fluid pressure determines whether the enhanced penetration and retention effect of nano-preparations can exist.

MRI-Based Interstitial Fluid Velocity Analysis for Drug Delivery Efficiency Evaluation in Tumor.

There are many flow behaviors in solid tumors, including intravascular, bloodstream, and interstitial convection. Studies have shown that tumor interstitial fluid (TIF) is an important part of tumor microenvironment regulation and affects drug delivery and metabolism between tumor cells. Magnetic resonance imaging (MRI) is suitable for detecting the flow rates of liquids in tissues. Clinical phase contrast PC-MRI technology has been designed to observe the blood flow in large vessels such as arteries and veins; however, it is not sensitive enough to deal with slow flow velocity. Our previously developed vertical plane echo PC-MRI technology, the Velocity Mapping sequence, improved the signal-to-noise ratio (SNR) for measuring slow interstitial fluid rate. In this study, this sequence was used to determine the TIF flow rate in MDA-MB-231 human breast tumor cells used in BALB/c nude male mice. Two different sizes of contrast agents were intravenously injected, and the relationship between their distribution and the TIF flow rate was studied for the first time. Combining the results of clinical scanning showed that small-molecule DTPA-Gd (diethylenetriaminepentaacetic acid-gadolinium) was distributed immediately around the tumor margin after the injection. This distribution was positively correlated to the high flow rate area of the TIF before administration. In contrast, nanoparticles NaGdF4-PEG (polyethylene glycol) entered the tumor and reached their peak at 3 h. Drug distribution was negatively correlated with the high-flow-rate region of the TIF. Investigation of the TIF velocity can help better understand the fluid behavior in tumors and its role in drug delivery.

Methodological and analytical considerations for intra-operative microdialysis.

BACKGROUND: Microdialysis is a technique that can be utilized to sample the interstitial fluid of the central nervous system (CNS), including in primary malignant brain tumors known as gliomas. Gliomas are mainly accessible at the time of surgery, but have rarely been analyzed via interstitial fluid collected via microdialysis. To that end, we obtained an investigational device exemption for high molecular weight catheters (HMW, 100 kDa) and a variable flow rate pump to perform microdialysis at flow rates amenable to an intra-operative setting. We herein report on the lessons and insights obtained during our intra-operative HMW microdialysis trial, both in regard to methodological and analytical considerations. METHODS: Intra-operative HMW microdialysis was performed during 15 clinically indicated glioma resections in fourteen patients, across three radiographically diverse regions in each patient. Microdialysates were analyzed via targeted and untargeted metabolomics via ultra-performance liquid chromatography tandem mass spectrometry. RESULTS: Use of albumin and lactate-containing perfusates impacted subsets of metabolites evaluated via global metabolomics. Additionally, focal delivery of lactate via a lactate-containing perfusate, induced local metabolic changes, suggesting the potential for intra-operative pharmacodynamic studies via reverse microdialysis of candidate drugs. Multiple peri-operatively administered drugs, including levetiracetam, cefazolin, caffeine, mannitol and acetaminophen, could be detected from one microdialysate aliquot representing 10 min worth of intra-operative sampling. Moreover, clinical, radiographic, and methodological considerations for performing intra-operative microdialysis are discussed. CONCLUSIONS: Intra-operative HMW microdialysis can feasibly be utilized to sample the live human CNS microenvironment, including both metabolites and drugs, within one surgery. Certain variables, such as perfusate type, must be considered during and after analysis. Trial registration NCT04047264.

Tumor interstitial fluid analysis enables the study of microenvironment-cell interactions in cancers.

The tumor microenvironment (TME) plays a crucial role in regulating the state and function of all cell types residing in the tumor and thus impacts many aspects of tumor biology. The importance of the TME has led to an interest in characterizing the composition of the TME and how TME components regulate cancer and stromal cell biology. Tumor interstitial fluid (TIF) is the local perfusate of the TME that carries metabolites, electrolytes, and soluble macromolecules to tumor-resident cells. Recently, techniques to isolate TIF have been coupled with analytical techniques to interrogate the composition of TIF, providing new insight into TME composition. In this review, we will discuss what TIF studies indicate about TME composition and new avenues TIF analysis provides to delineate how the TME regulates tumor biology.

Neural activity induces strongly coupled electro-chemo-mechanical interactions and fluid flow in astrocyte networks and extracellular space-A computational study.

The complex interplay between chemical, electrical, and mechanical factors is fundamental to the function and homeostasis of the brain, but the effect of electrochemical gradients on brain interstitial fluid flow, solute transport, and clearance remains poorly quantified. Here, via in-silico experiments based on biophysical modeling, we estimate water movement across astrocyte cell membranes, within astrocyte networks, and within the extracellular space (ECS) induced by neuronal activity, and quantify the relative role of different forces (osmotic, hydrostatic, and electrical) on transport and fluid flow under such conditions. We find that neuronal activity alone may induce intracellular fluid velocities in astrocyte networks of up to 14µm/min, and fluid velocities in the ECS of similar magnitude. These velocities are dominated by an osmotic contribution in the intracellular compartment; without it, the estimated fluid velocities drop by a factor of ×34-45. Further, the compartmental fluid flow has a pronounced effect on transport: advection accelerates ionic transport within astrocytic networks by a factor of ×1-5 compared to diffusion alone.

GABA promotes interstitial fluid clearance in an AQP4-dependent manner by activating the GABAA R.

The glymphatic system is a newly discovered perivascular network where cerebrospinal fluid mixes with interstitial fluid, facilitating clearance of protein solutes and metabolic waste from the parenchyma. The process is strictly dependent on water channel aquaporin-4 (AQP4) expressed on the perivascular astrocytic end-feet. Various factors, such as noradrenaline levels related to the arousal state, influence clearance efficiency, highlighting the possibility that other neurotransmitters additionally modulate this process. To date, the specific role of γ-aminobutyric acid (GABA) in the glymphatic system remains unknown. We used C57BL/6J mice to observe the regulatory effect of GABA on glymphatic pathway by administering a cerebrospinal fluid tracer containing GABA or its GABAA receptor (GABAA R) antagonist through cisterna magna injection. Then, we employed an AQP4 knockout mouse model to explore the regulatory effects of GABA on glymphatic drainage and further study whether transcranial magnetic stimulation-continuous theta burst stimulation (cTBS) could regulate the glymphatic pathway through the GABA system. Our data showed that GABA promotes glymphatic clearance in an AQP4-dependent manner by activating the GABAA R. Furthermore, cTBS was found to modulate the glymphatic pathway by activating the GABA system. Accordingly, we propose that regulating the GABA system by cTBS could modulate glymphatic clearance and provide new insight for clinical prevention and treatment of abnormal protein deposition-related diseases.

Microneedle-Integrated Sensors for Extraction of Skin Interstitial Fluid and Metabolic Analysis.

Skin interstitial fluid (ISF) has emerged as a fungible biofluid sample for blood serum and plasma for disease diagnosis and therapy. The sampling of skin ISF is highly desirable considering its easy accessibility, no damage to blood vessels, and reduced risk of infection. Particularly, skin ISF can be sampled using microneedle (MN)-based platforms in the skin tissues, which exhibit multiple advantages including minimal invasion of the skin tissues, less pain, ease of carrying, capacity for continuous monitoring, etc. In this review, we focus on the current development of microneedle-integrated transdermal sensors for collecting ISF and detecting specific disease biomarkers. Firstly, we discussed and classified microneedles according to their structural design, including solid MNs, hollow MNs, porous MNs, and coated MNs. Subsequently, we elaborate on the construction of MN-integrated sensors for metabolic analysis with highlights on the electrochemical, fluorescent, chemical chromogenic, immunodiagnostic, and molecular diagnostic MN-integrated sensors. Finally, we discuss the current challenges and future direction for developing MN-based platforms for ISF extraction and sensing applications.

Non-invasive imaging of interstitial fluid transport parameters in solid tumors in vivo.

In this paper, new and non-invasive imaging methods to assess interstitial fluid transport parameters in tumors in vivo are developed, analyzed and experimentally validated. These parameters include extracellular volume fraction (EVF), interstitial fluid volume fraction (IFVF) and interstitial hydraulic conductivity (IHC), and they are known to have a critical role in cancer progression and drug delivery effectiveness. EVF is defined as the volume of extracellular matrix per unit volume of the tumor, while IFVF refers to the volume of interstitial fluid per unit bulk volume of the tumor. There are currently no established imaging methods to assess interstitial fluid transport parameters in cancers in vivo. We develop and test new theoretical models and imaging techniques to assess fluid transport parameters in cancers using non-invasive ultrasound methods. EVF is estimated via the composite/mixture theory with the tumor being modeled as a biphasic (cellular phase and extracellular phase) composite material. IFVF is estimated by modeling the tumor as a biphasic poroelastic material with fully saturated solid phase. Finally, IHC is estimated from IFVF using the well-known Kozeny-Carman method inspired by soil mechanics theory. The proposed methods are tested using both controlled experiments and in vivo experiments on cancers. The controlled experiments were performed on tissue mimic polyacrylamide samples and validated using scanning electron microscopy (SEM). In vivo applicability of the proposed methods was demonstrated using a breast cancer model implanted in mice. Based on the controlled experimental validation, the proposed methods can estimate interstitial fluid transport parameters with an error below 10% with respect to benchmark SEM data. In vivo results demonstrate that EVF, IFVF and IHC increase in untreated tumors whereas these parameters are observed to decrease over time in treated tumors. The proposed non-invasive imaging methods may provide new and cost-effective diagnostic and prognostic tools to assess clinically relevant fluid transport parameters in cancers in vivo.

Controllable-Swelling Microneedle-Assisted Ultrasensitive Paper Sensing Platforms for Personal Health Monitoring.

Microneedle (MN) patches, which allow the extraction of skin interstitial fluid (ISF) without a pain sensation, are powerful tools for minimally invasive biofluid sampling. Herein, an MN-assisted paper-based sensing platform that enables rapid and painless biofluid analysis with ultrasensitive molecular recognition capacity is developed. First, a controllable-swelling MN patch is constructed through the engineering of a poly(ethylene glycol) diacrylate/methacrylated hyaluronic acid hydrogel; it combines rapid, sufficient extraction of ISF with excellent structural integrity. Notably, the analyte molecules in the needles can be recovered into a moist cellulose paper through spontaneous diffusion. More importantly, the paper can be functionalized with enzymatic colorimetric reagents or a plasmonic array, enabling a desired detection capacity-for example, the use of paper-based surface-enhanced Raman spectroscopy sensors leads to label-free, trace detection (sub-ppb level) of a diverse set of molecules (cefazolin, nicotine, paraquat, methylene blue). Finally, nicotine is selected as a model drug to evaluate the painless monitoring of three human volunteers. The changes in the nicotine levels can be tracked, with the levels varying significantly in response to the metabolism of drug in different volunteers. This as-designed minimally invasive sensing system should open up new opportunities for precision medicine, especially for personal healthcare monitoring.

Biomarkers Found in the Tumor Interstitial Fluid may Help Explain the Differential Behavior Among Keratinocyte Carcinomas.

Basal cell carcinomas (BCCs) and cutaneous squamous cell carcinomas (SCCs) are the most frequent types of cancer, and both originate from the keratinocyte transformation, giving rise to the group of tumors called keratinocyte carcinomas (KCs). The invasive behavior is different in each group of KC and may be influenced by their tumor microenvironment. The principal aim of the study is to characterize the protein profile of the tumor interstitial fluid (TIF) of KC to evaluate changes in the microenvironment that could be associated with their different invasive and metastatic capabilities. We obtained TIF from 27 skin biopsies and conducted a label-free quantitative proteomic analysis comparing seven BCCs, 16 SCCs, and four normal skins. A total of 2945 proteins were identified, 511 of them quantified in more than half of the samples of each tumoral type. The proteomic analysis revealed differentially expressed TIF proteins that could explain the different metastatic behavior in both KCs. In detail, the SCC samples disclosed an enrichment of proteins related to cytoskeleton, such as Stratafin and Ladinin-1. Previous studies found their upregulation positively correlated with tumor progression. Furthermore, the TIF of SCC samples was enriched with the cytokines S100A8/S100A9. These cytokines influence the metastatic output in other tumors through the activation of NF-kB signaling. According to this, we observed a significant increase in nuclear NF-kB subunit p65 in SCCs but not in BCCs. In addition, the TIF of both tumors was enriched with proteins involved in the immune response, highlighting the relevance of this process in the composition of the tumor environment. Thus, the comparison of the TIF composition of both KCs provides the discovery of a new set of differential biomarkers. Among them, secreted cytokines such as S100A9 may help explain the higher aggressiveness of SCCs, while Cornulin is a specific biomarker for BCCs. Finally, the proteomic landscape of TIF provides key information on tumor growth and metastasis, which can contribute to the identification of clinically applicable biomarkers that may be used in the diagnosis of KC, as well as therapeutic targets.

Loss of aquaporin-4 results in glymphatic system dysfunction via brain-wide interstitial fluid stagnation.

The glymphatic system is a fluid transport network of cerebrospinal fluid (CSF) entering the brain along arterial perivascular spaces, exchanging with interstitial fluid (ISF), ultimately establishing directional clearance of interstitial solutes. CSF transport is facilitated by the expression of aquaporin-4 (AQP4) water channels on the perivascular endfeet of astrocytes. Mice with genetic deletion of AQP4 (AQP4 KO) exhibit abnormalities in the brain structure and molecular water transport. Yet, no studies have systematically examined how these abnormalities in structure and water transport correlate with glymphatic function. Here, we used high-resolution 3D magnetic resonance (MR) non-contrast cisternography, diffusion-weighted MR imaging (MR-DWI) along with intravoxel-incoherent motion (IVIM) DWI, while evaluating glymphatic function using a standard dynamic contrast-enhanced MR imaging to better understand how water transport and glymphatic function is disrupted after genetic deletion of AQP4. AQP4 KO mice had larger interstitial spaces and total brain volumes resulting in higher water content and reduced CSF space volumes, despite similar CSF production rates and vascular density compared to wildtype mice. The larger interstitial fluid volume likely resulted in increased slow but not fast MR diffusion measures and coincided with reduced glymphatic influx. This markedly altered brain fluid transport in AQP4 KO mice may result from a reduction in glymphatic clearance, leading to enlargement and stagnation of fluid in the interstitial space. Overall, diffusion MR is a useful tool to evaluate glymphatic function and may serve as valuable translational biomarker to study glymphatics in human disease.