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

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

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.

The extracellular space (ECS) is the network of interconnected channels exterior to all brain cells and contains both interstitial fluid and extracellular matrix (Figure 1a and Figure 1b). The distribution of many substances required for brain cell function, including nutrients, hormones, and neurotransmitters, occurs by diffusion through the ECS. Changes in the physical parameters of this space, including volume, geometry, and extracellular matrix, can drastically affect diffusion through the ECS and the local ion concentrations bathing brain cells, which have a profound impact on brain cell function

All animal procedures, used to obtain tissue samples, were approval by the animal ethics committee at SUNY Downstate Medical Center.

1. Preparation of Solutions and Equipment

1. Prepare a 150 mM NaCl backfill solution for the reference barrel of the ISM. Store it in a 10 mL syringe attached to a 0.22 µm filter (to remove bacteria or particles).
2. Prepare a 150 mM TMA chloride (TMA-Cl) backfill solution for the microelectrodes. Store it in a 10 mL syringe attached to a 0.2.......

The utility of the RTI technique is demonstrated in an experiment designed to measure the changes in α and during a hypoosmolar challenge (Figure 8 and Figure 9). It has previously been shown that reducing the osmolarity of the ECS by washing on hypotonic ACSF will produce a decrease in α and an increase in λ¹³.

Figure 10: Non-ideal Data Demonstrating Common Technical Issues. (a) Diagrams of common technical issues with iontophoresis microelectrodes: Comparison of the normal release of TMA from a functioning iontophoresis microelectrode with three sources demonstrating technical issues. [High magnification, a1] The current in an ideal iontophoretic source is carried equally by TMA release and chloride upta.......

The work was supported by NIH NINDS grant R01 NS047557.