Rapid volume pulsations of the extracellular space accompany epileptiform activity in trauma-injured neocortex and depend on the sodium-bicarbonate cotransporter NBCe1.

Post traumatic epilepsy (PTE) is a treatment-resistant consequence of traumatic brain injury (TBI). Recently, it has been revealed that epileptiform activity in acute chemoconvulsant seizure models is accompanied by transient shrinkages of extracellular space (ECS) called rapid volume pulsations (RVPs). Shrinkage of the ECS surrounding neurons and glia may contribute to ictogenic hyperexcitability and hypersynchrony during the chronic phase of TBI. Here, we identify the phenomenon of RVPs occurring spontaneously in rat neocortex at ≥ 3 weeks after injury in the controlled cortical impact (CCI) model for PTE. We further report that blocking the electrogenic action of the astrocytic cotransporter NBCe1 with 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminates both RVPs and epileptiform activity in ex-vivo CCI neocortical brain slices. We conclude that NBCe1-mediated extracellular volume shrinkage may represent a new target for therapeutic intervention in PTE.

BubbleDrive, a low-volume incubation chamber for acute brain slices.

Acute brain slices are a common and useful preparation in experimental neuroscience. A wide range of incubation chambers for brain slices exists but only a few are designed with very low volumes of the bath solution in mind. Such chambers are necessary when high-cost chemicals are to be added to the solution or when small amounts of substances released by the slice are to be collected for analysis. The principal challenge in designing a very low-volume incubation chamber is maintaining good oxygenation and flow without mechanically disturbing or damaging the slices. We designed and validated BubbleDrive, a 3D-printed incubation chamber with a minimum volume of 1.5 mL which can hold up to three coronal mouse slices from one hemisphere. It employs the carbogen gas bubbles to drive the flow circulation in a consistent and reproducible manner, and without disturbing the brain slices. The BubbleDrive design and construction were successfully validated by comparison to a conventional large-volume incubation chamber in several experimental designs involving measurements of extracellular diffusion parameters, the electrophysiology of neuronal and astrocytic networks, and the effectiveness of slice incubation with hyaluronidase enzyme.

Local diffusion in the extracellular space of the brain.

The brain extracellular space (ECS) is a vast interstitial reticulum of extreme morphological complexity, composed of narrow gaps separated by local expansions, enabling interconnected highways between neural cells. Constituting on average 20% of brain volume, the ECS is key for intercellular communication, and understanding its diffusional properties is of paramount importance for understanding the brain. Within the ECS, neuroactive substances travel predominantly by diffusion, spreading through the interstitial fluid and the extracellular matrix scaffold after being focally released. The nanoscale dimensions of the ECS render it unresolvable by conventional live tissue compatible imaging methods, and historically diffusion of tracers has been used to indirectly infer its structure. Novel nanoscopic imaging techniques now show that the ECS is a highly dynamic compartment, and that diffusivity in the ECS is more heterogeneous than anticipated, with great variability across brain regions and physiological states. Diffusion is defined primarily by the local ECS geometry, and secondarily by the viscosity of the interstitial fluid, including the obstructive and binding properties of the extracellular matrix. ECS volume fraction and tortuosity both strongly determine diffusivity, and each can be independently regulated e.g. through alterations in glial morphology and the extracellular matrix composition. Here we aim to provide an overview of our current understanding of the ECS and its diffusional properties. We highlight emerging technological advances to respectively interrogate and model diffusion through the ECS, and point out how these may contribute in resolving the remaining enigmas of the ECS.

The glymphatic system: Current understanding and modeling.

We review theoretical and numerical models of the glymphatic system, which circulates cerebrospinal fluid and interstitial fluid around the brain, facilitating solute transport. Models enable hypothesis development and predictions of transport, with clinical applications including drug delivery, stroke, cardiac arrest, and neurodegenerative disorders like Alzheimer's disease. We sort existing models into broad categories by anatomical function: Perivascular flow, transport in brain parenchyma, interfaces to perivascular spaces, efflux routes, and links to neuronal activity. Needs and opportunities for future work are highlighted wherever possible; new models, expanded models, and novel experiments to inform models could all have tremendous value for advancing the field.

Multiscale Computer Modeling of Spreading Depolarization in Brain Slices.

Spreading depolarization (SD) is a slow-moving wave of neuronal depolarization accompanied by a breakdown of ion concentration homeostasis, followed by long periods of neuronal silence (spreading depression), and is associated with several neurologic conditions. We developed multiscale (ions to tissue slice) computer models of SD in brain slices using the NEURON simulator: 36,000 neurons (two voltage-gated ion channels; three leak channels; three ion exchangers/pumps) in the extracellular space (ECS) of a slice (1 mm sides, varying thicknesses) with ion (K+, Cl-, Na+) and O2 diffusion and equilibration with a surrounding bath. Glia and neurons cleared K+ from the ECS via Na+/K+ pumps. SD propagated through the slices at realistic speeds of 2-4 mm/min, which increased by as much as 50% in models incorporating the effects of hypoxia or propionate. In both cases, the speedup was mediated principally by ECS shrinkage. Our model allows us to make testable predictions, including the following: (1) SD can be inhibited by enlarging ECS volume; (2) SD velocity will be greater in areas with greater neuronal density, total neuronal volume, or larger/more dendrites; (3) SD is all-or-none: initiating K+ bolus properties have little impact on SD speed; (4) Slice thickness influences SD because of relative hypoxia in the slice core, exacerbated by SD in a pathologic cycle; and (5) SD and high neuronal spike rates will be observed in the core of the slice. Cells in the periphery of the slice near an oxygenated bath will resist SD.

Probing Neuropeptide Volume Transmission In Vivo by Simultaneous Near-Infrared Light-Triggered Release and Optical Sensing.

Neuropeptides are abundant signaling molecules in the central nervous system. Yet remarkably little is known about their spatiotemporal spread and biological activity. Here, we developed an integrated optical approach using Plasmonic nAnovesicles and cell-based neurotransmitter fluorescent engineered reporter (CNiFER), or PACE, to probe neuropeptide signaling in the mouse neocortex. Small volumes (fL to pL) of exogenously supplied somatostatin-14 (SST) can be rapidly released under near-infrared light stimulation from nanovesicles implanted in the brain and detected by SST2 CNiFERs with nM sensitivity. Our measurements reveal reduced but synchronized SST transmission within 130 µm, and markedly smaller and delayed transmission at longer distances. These measurements enabled a quantitative estimation of the SST loss rate due to peptide degradation and binding. PACE offers a new tool for determining the spatiotemporal scales of neuropeptide volume transmission and signaling in the brain.

Brain extracellular space of the naked mole-rat expands and maintains normal diffusion under ischemic conditions.

Brain extracellular space (ECS) forms a conduit for diffusion, an essential mode of molecular transport between brain vasculature, neurons and glia. ECS volume is reduced under conditions of hypoxia and ischemia, contributing to impaired extracellular diffusion and consequent neuronal dysfunction and death. We investigated the ECS volume fraction and diffusion permeability of the African naked mole-rat (NM-R; Heterocephalus Glaber), a rodent with a remarkably high tolerance for hypoxia and ischemia. Real-Time Iontophoretic and Integrative Optical Imaging methods were used to evaluate diffusion transport in cortical slices under normoxic and ischemic conditions, and results were compared to values previously collected in rats. NM-R brains under normoxic conditions had a smaller ECS volume fraction than rats, and a correspondingly decreased diffusion permeability for macromolecules. Surprisingly, and in sharp contrast to rats, the NM-R ECS responded to ischemic conditions at the center of thick brain slices by expanding, rather than shrinking, and preserving diffusion permeabilities for small and large molecules. The NM-R thick slices also showed a blunted accumulation of ECS potassium compared to rats. The remarkable dynamic response of the NM-R ECS to ischemia likely demonstrates an adaptation for NM-R to maintain brain function in their extreme nest environment.

Rapid volume pulsation of the extracellular space coincides with epileptiform activity in mice and depends on the NBCe1 transporter.

KEY POINTS: Extracellular space (ECS) rapid volume pulsation (RVP) accompanying epileptiform activity is described for the first time. Such RVP occurs robustly in several in vitro and in vivo mouse models of epileptiform activity. In the in vitro 4-aminopyridine model of epileptiform activity, RVP depends on the activity of the electrogenic Na+ /HCO3- cotransporter (NBCe1). NBCe1 pharmacological inhibition suppresses RVP and epileptiform activity. Inhibition of changes in ECS volume may be a useful target in epilepsy patients who are resistant to current treatments. ​ ABSTRACT: The extracellular space (ECS) of the brain shrinks persistently by approximately 35% during epileptic seizures. Here we report the discovery of rapid volume pulsation (RVP), further transient drops in ECS volume which accompany events of epileptiform activity. These transient ECS contractions were observed in multiple mouse models of epileptiform activity both in vivo (bicuculline methiodide model) and in vitro (hyaluronan synthase 3 knock-out, picrotoxin, bicuculline and 4-aminopyridine models). By using the probe transients quantification (PTQ) method we show that individual pulses of RVP shrank the ECS by almost 15% in vivo. In the 4-aminopyridine in vitro model, the individual pulses of RVP shrank the ECS by more than 4%, and these transient changes were superimposed on a persistent ECS shrinkage of 36% measured with the real-time iontophoretic method. In this in vitro model, we investigated several channels and transporters that may be required for the generation of RVP and epileptiform activity. Pharmacological blockages of Na+ /K+ /2Cl- cotransporter type 1 (NKCC1), K+ /Cl- cotransporter (KCC2), the water channel aquaporin-4 (AQP4) and inwardly rectifying potassium channel 4.1 (Kir4.1) were ineffective in halting the RVP and the epileptiform activity. In contrast, pharmacological blockade of the electrogenic Na+ /HCO3- cotransporter (NBCe1) by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) eliminated both the RVP and the persistent ECS shrinkage. Importantly, this blocker also stopped the epileptiform activity. These results demonstrate that RVP is closely associated with epileptiform activity across several models of epileptiform activity and therefore the underlying mechanism could potentially represent a novel target for epilepsy management and treatment.

Integrity of White Matter is Compromised in Mice with Hyaluronan Deficiency.

Brain white matter is the means of efficient signal propagation in brain and its dysfunction is associated with many neurological disorders. We studied the effect of hyaluronan deficiency on the integrity of myelin in murine corpus callosum. Conditional knockout mice lacking the hyaluronan synthase 2 were compared with control mice. Ultrastructural analysis by electron microscopy revealed a higher proportion of myelin lamellae intruding into axons of knockout mice, along with significantly slimmer axons (excluding myelin sheath thickness), lower g-ratios, and frequent loosening of the myelin wrappings, even though the myelin thickness was similar across the genotypes. Analysis of extracellular diffusion of a small marker molecule tetramethylammonium (74 MW) in brain slices prepared from corpus callosum showed that the extracellular space volume increased significantly in the knockout animals. Despite this vastly enlarged volume, extracellular diffusion rates were significantly reduced, indicating that the compromised myelin wrappings expose more complex geometric structure than the healthy ones. This finding was confirmed in vivo by diffusion-weighted magnetic resonance imaging. Magnetic resonance spectroscopy suggested that water was released from within the myelin sheaths. Our results indicate that hyaluronan is essential for the correct formation of tight myelin wrappings around the axons in white matter.

Time-Resolved Integrative Optical Imaging of Diffusion during Spreading Depression.

An improved version of the integrative optical imaging method has been developed that substantially increases the time resolution of diffusion measurements. We present a theory for time-resolved integrative optical imaging that incorporates a time-dependent effective diffusion coefficient in homogeneous anisotropic media and a time-dependent nonspecific linear clearance. The method was applied to measure the very fast changes in extracellular diffusion that occur during spreading depression in rat hippocampal slices. We were able to achieve a time resolution of approximately 1 s, an improvement of at least 10 times compared to the standard methods for extracellular diffusion measurement. We have found that diffusion of a small fluorescent extracellular marker (MW 3000) completely stopped during the maximum direct current shift associated with the spreading depression wave, then gradually resumed over several minutes afterward. The effect of spreading depression on extracellular space is much larger than previously estimated by other methods with lower time resolution.

ECS Dynamism and Its Influence on Neuronal Excitability and Seizures.

Seizure activity is governed by changes in normal neuronal physiology that lead to a state of neuronal hyperexcitability and synchrony. There is a growing body of research and evidence suggesting that alterations in the volume fraction (α) of the brain's extracellular space (ECS) have the ability to prolong or even initiate seizures. These ictogenic effects likely occur due to the ECS volume being critically important in determining both the concentration of neuroactive substances contained within it, such as ions and neurotransmitters, and the effect of electric field-mediated interactions between neurons. Changes in the size of the ECS likely both precede a seizure, assisting in its initiation, and occur during a seizure, assisting in its maintenance. Different cellular ion and water transporters and channels are essential mediators in determining neuronal excitability and synchrony and can do so through alterations in ECS volume and/or through non-ECS volume related mechanisms. This review will parse out the relationships between how the ECS volume changes during normal physiology and seizures, how those changes might alter neuronal physiology to promote seizures, and what ion and water transporters and channels are important in linking ECS volume changes and seizures.

Unveiling the Extracellular Space of the Brain: From Super-resolved Microstructure to In Vivo Function.

The extracellular space occupies approximately one-fifth of brain volume, molding a spider web of gaps filled with interstitial fluid and extracellular matrix where neurons and glial cells perform in concert. Yet, very little is known about the spatial organization and dynamics of the extracellular space, let alone its influence on brain function, owing to a lack of appropriate techniques (and a traditional bias toward the inside of cells, not the spaces in between). At the same time, it is clear that understanding fundamental brain functions, such as synaptic transmission, memory, sleep, and recovery from disease, calls for more focused research on the extracellular space of the brain. This review article highlights several key research areas, covering recent methodological and conceptual progress that illuminates this understudied, yet critically important, brain compartment, providing insights into the opportunities and challenges of this nascent field.

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space.

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 µm away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

Brain extracellular space, hyaluronan, and the prevention of epileptic seizures.

Mutant mice deficient in hyaluronan (HA) have an epileptic phenotype. HA is one of the major constituents of the brain extracellular matrix. HA has a remarkable hydration capacity, and a lack of HA causes reduced extracellular space (ECS) volume in the brain. Reducing ECS volume can initiate or exacerbate epileptiform activity in many in vitro models of epilepsy. There is both in vitro and in vivo evidence of a positive feedback loop between reduced ECS volume and synchronous neuronal activity. Reduced ECS volume promotes epileptiform activity primarily via enhanced ephaptic interactions and increased extracellular potassium concentration; however, the epileptiform activity in many models, including the brain slices from HA synthase-3 knockout mice, may still require glutamate-mediated synaptic activity. In brain slice epilepsy models, hyperosmotic solution can effectively shrink cells and thus increase ECS volume and block epileptiform activity. However, in vivo, the intravenous administration of hyperosmotic solution shrinks both brain cells and brain ECS volume. Instead, manipulations that increase the synthesis of high-molecular-weight HA or decrease its breakdown may be used in the future to increase brain ECS volume and prevent seizures in patients with epilepsy. The prevention of epileptogenesis is also a future target of HA manipulation. Head trauma, ischemic stroke, and other brain insults that initiate epileptogenesis are known to be associated with an early decrease in high-molecular-weight HA, and preventing that decrease in HA may prevent the epileptogenesis.

Brain Extracellular Space: The Final Frontier of Neuroscience.

Brain extracellular space is the narrow microenvironment that surrounds every cell of the central nervous system. It contains a solution that closely resembles cerebrospinal fluid with the addition of extracellular matrix molecules. The space provides a reservoir for ions essential to the electrical activity of neurons and forms an intercellular chemical communication channel. Attempts to reveal the size and structure of the extracellular space using electron microscopy have had limited success; however, a biophysical approach based on diffusion of selected probe molecules has proved useful. A point-source paradigm, realized in the real-time iontophoresis method using tetramethylammonium, as well as earlier radiotracer methods, have shown that the extracellular space occupies ∼20% of brain tissue and small molecules have an effective diffusion coefficient that is two-fifths that in a free solution. Monte Carlo modeling indicates that geometrical constraints, including dead-space microdomains, contribute to the hindrance to diffusion. Imaging the spread of macromolecules shows them increasingly hindered as a function of size and suggests that the gaps between cells are predominantly ∼40 nm with wider local expansions that may represent dead-spaces. Diffusion measurements also characterize interactions of ions and proteins with the chondroitin and heparan sulfate components of the extracellular matrix; however, the many roles of the matrix are only starting to become apparent. The existence and magnitude of bulk flow and the so-called glymphatic system are topics of current interest and controversy. The extracellular space is an exciting area for research that will be propelled by emerging technologies.

Activation of ß-adrenergic receptors in rat visual cortex expands astrocytic processes and reduces extracellular space volume.

Brain extracellular space (ECS) is an interconnected channel that allows diffusion-mediated transport of signaling molecules, metabolites, and drugs. We tested the hypothesis that ß-adrenergic receptor (ßAR) activation impacts extracellular diffusion-mediated transport of molecules through alterations in the morphology of astrocytes. Two structural parameters of ECS-volume fraction and tortuosity-govern extracellular diffusion. Volume fraction (α) is the volume of ECS relative to the total tissue volume. Tortuosity (λ) is a measure of the hindrance that molecules experience in the ECS, compared to a free medium. The real-time iontophoretic (RTI) method revealed that treatment of acutely prepared visual cortical slices of adult female rats with a ßAR agonist, DL-isoproterenol (ISO), decreases α significantly, from 0.22 ± 0.03 (mean ± SD) for controls without agonist to 0.18 ± 0.03 with ISO, without altering λ (control: 1.64 ± 0.04; ISO: 1.63 ± 0.04). Electron microscopy revealed that the ISO treatment significantly increased the cytoplasmic area of astrocytic distal endings per unit area of neuropil by 54%. These findings show that norepinephrine decreases α, in part, through an increase in astrocytic volume following ßAR activation. Norepinephrine is recognized to be released within the brain during the awake state and increase neurons' signal-to-noise ratio through modulation of neurons' biophysical properties. Our findings uncover a new mechanism for noradrenergic modulation of neuronal signals. Through astrocytic activation leading to a reduction of α, noradrenergic modulation increases extracellular concentration of neurotransmitters and neuromodulators, thereby facilitating neuronal interactions, especially during wakefulness. Synapse 70:307-316, 2016. © 2016 Wiley Periodicals, Inc.

T-type calcium channels contribute to calcium disturbances in brain during hyponatremia.

Disturbance of calcium homeostasis is implicated in the normal process of aging and brain pathology prevalent in the elderly such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. Previous studies demonstrated that applying a hyponatremic iso-osmotic (low-NaCl) artificial cerebrospinal fluid (ACSF) to rodent hippocampus causes extracellular calcium to rapidly decrease. Restoring normonatremia after low-NaCl treatment causes a rapid increase in extracellular calcium that overshoots baseline. This study examined the amplitude, timing, and mechanism of these surprising calcium changes. We also tested whether hyponatremia increased calcium entry into brain cells or calcium binding to chondroitin sulfate (CS), a negatively charged constituent of the extracellular matrix (ECM) that may be occupied by sodium during normonatremia. We report three major findings. First we show that CS does not contribute to extracellular calcium changes during low-NaCl treatments. Second, we show that the time to minimum extracellular calcium during low-NaCl treatment is significantly shorter than the time to maximum extracellular calcium in recovery from low-NaCl treatment. Third, we show that the decrease in extracellular calcium observed during hyponatremia is attenuated by ML 218, a highly selective T-type calcium channel blocker. Together these data suggest that calcium rapidly enters cells at the onset of low-NaCl treatment and is extruded from cells when normonatremia is restored. Calcium binding to CS does not significantly contribute to calcium changes in brain during hyponatremia. Differences in timing suggest that extracellular calcium changes during and in recovery from hyponatremia occur by distinct mechanisms or by a multistep process. Finally, partial block of extracellular calcium influx by ML 218 suggests that T-type channels are involved in calcium entering cells during hyponatremia. Given the high prevalence of hyponatremia among elderly patients and the growing understanding of calcium's role in multiple neurologic pathologies, this study promotes a novel approach for studying and potentially preventing the effects of hyponatremia on calcium dysregulation in brain tissue.

Anomalous extracellular diffusion in rat cerebellum.

Extracellular space (ECS) is a major channel transporting biologically active molecules and drugs in the brain. Diffusion-mediated transport of these substances is hindered by the ECS structure but the microscopic basis of this hindrance is not fully understood. One hypothesis proposes that the hindrance originates in large part from the presence of dead-space (DS) microdomains that can transiently retain diffusing molecules. Because previous theoretical and modeling work reported an initial period of anomalous diffusion in similar environments, we expected that brain regions densely populated by DS microdomains would exhibit anomalous extracellular diffusion. Specifically, we targeted granular layers (GL) of rat and turtle cerebella that are populated with large and geometrically complex glomeruli. The integrative optical imaging (IOI) method was employed to evaluate diffusion of fluorophore-labeled dextran (MW 3000) in GL, and the IOI data analysis was adapted to quantify the anomalous diffusion exponent dw from the IOI records. Diffusion was significantly anomalous in rat GL, where dw reached 4.8. In the geometrically simpler turtle GL, dw was elevated but not robustly anomalous (dw = 2.6). The experimental work was complemented by numerical Monte Carlo simulations of anomalous ECS diffusion in several three-dimensional tissue models containing glomeruli-like structures. It demonstrated that both the duration of transiently anomalous diffusion and the anomalous exponent depend on the size of model glomeruli and the degree of their wrapping. In conclusion, we have found anomalous extracellular diffusion in the GL of rat cerebellum. This finding lends support to the DS microdomain hypothesis. Transiently anomalous diffusion also has a profound effect on the spatiotemporal distribution of molecules released into the ECS, especially at diffusion distances on the order of a few cell diameters, speeding up short-range diffusion-mediated signals in less permeable structures.

Hyaluronan deficiency due to Has3 knock-out causes altered neuronal activity and seizures via reduction in brain extracellular space.

Hyaluronan (HA), a large anionic polysaccharide (glycosaminoglycan), is a major constituent of the extracellular matrix of the adult brain. To address its function, we examined the neurophysiology of knock-out mice deficient in hyaluronan synthase (Has) genes. Here we report that these Has mutant mice are prone to epileptic seizures, and that in Has3(-/-) mice, this phenotype is likely derived from a reduction in the size of the brain extracellular space (ECS). Among the three Has knock-out models, namely Has3(-/-), Has1(-/-), and Has2(CKO), the seizures were most prevalent in Has3(-/-) mice, which also showed the greatest HA reduction in the hippocampus. Electrophysiology in Has3(-/-) brain slices demonstrated spontaneous epileptiform activity in CA1 pyramidal neurons, while histological analysis revealed an increase in cell packing in the CA1 stratum pyramidale. Imaging of the diffusion of a fluorescent marker revealed that the transit of molecules through the ECS of this layer was reduced. Quantitative analysis of ECS by the real-time iontophoretic method demonstrated that ECS volume was selectively reduced in the stratum pyramidale by ∼ 40% in Has3(-/-) mice. Finally, osmotic manipulation experiments in brain slices from Has3(-/-) and wild-type mice provided evidence for a causal link between ECS volume and epileptiform activity. Our results provide the first direct evidence for the physiological role of HA in the regulation of ECS volume, and suggest that HA-based preservation of ECS volume may offer a novel avenue for development of antiepileptogenic treatments.