The long-term goal of our research is to construct and characterize a realistic three-dimensional model of the brain extracellular space (ECS), in order to predict the impact of microstructural changes on the transport of signaling molecules, nutrients and therapeutic agents. ECS comprises the narrow channels that separate brain cells but cannot be directly visualized in the living brain. It is essential for normal brain function and influences many critical processes including intercellular signaling, nutrient delivery and neurotrophic effects. Significantly, the ECS also forms the final route for all drug delivery to brain cells. To develop quantitative understanding of any of these diffusion mediated processes, essential structural parameters of the complex ECS environment must be identified and characterized. Traditional diffusion measurements, made over large distances and long times, extract two macroscopic parameters, volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by the ECS, and tortuosity quantifies average hindrance imposed on diffusing molecules by the complex ECS environment. Both parameters are affected by ECS manipulations, pathological conditions, and cellular activity. However, interpretation of the traditional macroscopic diffusion experiments in terms of the microscopic tissue properties is difficult and ambiguous. The principal goal of this proposal is to develop and deploy diffusion measurements with much higher spatial and time resolutions, in order to better match the ECS microstructure.
In Aim 1, we propose to develop a new diffusion method with spatial resolution improved by a factor of 10 compared to the traditional methods. Preliminary high-resolution experiments document transient anomalous diffusion on a scale of tens of micrometers. Diffusion stabilizes only after the molecules fully experience the complexity of the ECS environment. We call the smallest volume containing all of the ECS structural complexity a Dynamic Microdomain (DM). The size of the DM represents an important new tissue parameter, which will be measured and evaluated in addition to the traditional tortuosity and volume fraction.
Aims 2 −4 explore important aspects of the DMs.
Aim 2 examines how the beta2-adrenergic signaling invokes glia plasticity and alters the DM diffusion properties, which ultimately leads to modulation of neuronal excitability and signaling.
Aim 3 tests the hypothesis that perineuronal nets function as charge discriminating components within the DMs. Perineuronal nets, formed by negatively charged glycans of extracellular matrix, are found close to neurons in specialized ECS regions surrounded by glia. We propose that this high negative charge density attracts polyvalent cations but repulses polyvalent anions.
Aim 4 will establish the average width of the spaces separating the cells. Very few estimates of this basic parameter exist in a living cortical tissue. The scaling theory of polymer diffusion predicts the pore width from the diameter of a flexible polymer compelled to diffuse in the reptation regime. The characteristic pore width is essential for any realistic model of the DM and ECS, and equally so for construction of efficient drug carriers.

Public Health Relevance

Brain cells, comprising neurons and glia, are surrounded by extracellular space (ECS), a system of interconnected pores that channels chemical signals between cells and is an essential route for delivery of nutrients and drugs. This project combines experiments to measure diffusion in brain tissue with mathematical modeling to characterize the microstructure of the ECS and how it is regulated. The results will be important both for understanding how altered ECS structure in neuropathological states disrupts the chemical traffic of the brain, and for designing effective strategies to deliver drugs in patients suffering from neurological disorders and brain tumors.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
High Priority, Short Term Project Award (R56)
Project #
Application #
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Jacobs, Tom P
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Suny Downstate Medical Center
Anatomy/Cell Biology
Schools of Medicine
United States
Zip Code
Sherpa, Ang Doma; Xiao, Fanrong; Joseph, Neethu et al. (2016) Activation of ?-adrenergic receptors in rat visual cortex expands astrocytic processes and reduces extracellular space volume. Synapse 70:307-16
Sabaliauskas, Nicole; Shen, Hui; Molla, Jonela et al. (2015) Neurosteroid effects at ?4?? GABAA receptors alter spatial learning and synaptic plasticity in CA1 hippocampus across the estrous cycle of the mouse. Brain Res 1621:170-86
Xiao, Fanrong; Hrabe, Jan; Hrabetova, Sabina (2015) Anomalous extracellular diffusion in rat cerebellum. Biophys J 108:2384-95
Arranz, Amaia M; Perkins, Katherine L; Irie, Fumitoshi et al. (2014) Hyaluronan deficiency due to Has3 knock-out causes altered neuronal activity and seizures via reduction in brain extracellular space. J Neurosci 34:6164-76
Sherpa, Ang Doma; van de Nes, Paula; Xiao, Fanrong et al. (2014) Gliotoxin-induced swelling of astrocytes hinders diffusion in brain extracellular space via formation of dead-space microdomains. Glia 62:1053-65
Saghyan, Aleksandr; Lewis, David P; Hrabe, Jan et al. (2012) Extracellular diffusion in laminar brain structures exemplified by hippocampus. J Neurosci Methods 205:110-8
Xiao, F; Hrabetov√°, S (2009) Enlarged extracellular space of aquaporin-4-deficient mice does not enhance diffusion of Alexa Fluor 488 or dextran polymers. Neuroscience 161:39-45
Hrabetov√°, Sabina; Masri, Daniel; Tao, Lian et al. (2009) Calcium diffusion enhanced after cleavage of negatively charged components of brain extracellular matrix by chondroitinase ABC. J Physiol 587:4029-49