Imaging method measures width of brain’s extracellular space
Space may be larger than previously thought
Drugs and drug carriers targeting the brain can range from less than one to several hundred
nanometers in size and must travel through the brain tissue before being directed
to target sites. Knowing the structure and width of the brain tissue’s extracellular
space could help scientists design effective drug treatment strategies.
Previous electron microscopy experiments have
suggested that the brain’s extracellular space is between 10 and 20 nm wide
— obviously too small for drugs larger than 20 nm to maneuver around. However,
electron micrographs are indicative only of the space size after death —
when the brain is no longer functioning and when water has left the extracellular
Researchers have found more information about the width of the brain’s extracellular space.
The first image shows the cranial window of a rat’s skull under a fixed-stage
microscope. The second shows the cranial window just after a micropipette has been
lowered 200 μm into the brain. The last two fluorescence images show the micropipette
before and just after injection of 3-nm-wide fluorescent dextran into the brain
Robert G. Thorne and Charles Nicholson
from the department of physiology and neuroscience at New York University School
of Medicine decided to try studying diffusion through the extracellular space of
rats to see if a substance larger than 20 nm could diffuse through living brain
tissue. They found that extracellular space may be larger than once thought.
They experimented with integrative
optical imaging, a method previously developed by Nicholson and colleagues for diffusion
measurements in brain slices. It involves using epifluorescence microscopy with
quantitative image analysis to measure the diffusion of fluorescent probes after
their 50- to 200-ms pressure injection from a micropipette.
These images show the rate of diffusion
in the rat brain of the 3-nm-wide fluorescent dextran at 0, 13, 26 and 39 s.
As reported in the April 4 issue of
PNAS, the researchers decided to try adapting the method for in vivo use
to see if they could observe the diffusion of three fluorescent probes in living
rat brain tissue. Thorne said that minimizing movement in the brain tissue, because
the animal was alive, was the hardest part in their adaptation.
They first created an open cranial
window for observation in anesthetized rats by drilling away a small section of
the skull and carefully removing the dura mater. Then, using a micropipette, they
pressure-injected 3-nm-wide fluorescent dextran 200 μm into the brain. They
performed the same procedure (using different rat brains) with a 14-nm-wide fluorescent
dextran and then a 35-nm-wide CdSe quantum dot conjugate (polyethylene glycol-coated
to make it relatively inert. The three probes were chosen for their inertness to
minimize charge-based interactions with brain extracellular space components.
After a one- to two-hour equilibration
period following the surgery for the open cranial window, the researchers transferred
the rats to a fixed-stage microscope with a 0.3-NA water-immersion objective from
Olympus America Inc. of Melville, N.Y., a 75-W xenon epi-illuminator and a dichroic
mirror system to view the fluorescent probes.
They collected images in the brain
every 10 to 120 s after pressure-injection, using a CCD camera from Photometrics
of Tucson, Ariz. Image files were processed for diffusion analysis using software
from Digital Optics of Charlotte, N.C., running under a program written by Nicholson
in Matlab, developed by The MathWorks Inc. of Natick, Mass.
Thorne explained that, in theory, a
limit for the extracellular space width could be established by just injecting increasingly
larger probes into the brain tissue until diffusion stopped. “But you can
imagine, since this is a living animal, and we inject such a small volume of fluorescent
probes, observing diffusion in the animal for a long time is no trivial matter.”
Thorne said that the larger probes
took a long time to diffuse, with the 35-nm quantum dots taking nearly an hour to
spread an appreciable distance. So instead of watching hundreds of different probes,
the scientists observed three and applied restricted diffusion theory to estimate
from their data how large the extracellular space might be.
The restricted diffusion theory is
a hydrodynamic theory for hindered diffusion. It involves calculating the amount
of tortuosity (hindrance) the probes face from the tissue’s geometry, and
the chemicals and obstacles the probes come into contact with along their journey.
The tortuosity and the probe’s apparent diameter (determined by measuring
its diffusion in water) are then used to derive an estimate of the size of the extracellular
As expected, the researchers found
that the time required for diffusion was related to the probe’s size. The
3-nm-wide dextran took the least amount of time to diffuse, and the 35-nm-wide quantum
dot took the most. “The fact that the quantum dots diffused at all told us
that, at minimum, these spaces have to be at least 35 nm in size,” Thorne
Based on estimates obtained by applying
restricted diffusion theory, they found the extracellular space to be between 38
and 64 nm wide. “This is two to six times larger than historical estimates
based on electron microscopy measurements,” he said.
But the researchers didn’t stop
there. They also studied the size of the space after terminal ischemia, a condition
that rapidly subjects the brain to energy depletion after cardiac arrest. They did
so to compare findings from previous research using electron micrographs of brain
tissues that had experienced terminal ischemia with their restricted diffusion theory
estimates using integrative optical imaging.
The investigators observed the 3-nm-wide
probe’s diffusion after inducing immediate cardiac arrest and terminal ischemia
by injecting potassium chloride into the animals. “We found the size of the
extracellular space shrunk to less than 10 nm wide after just a couple of minutes,”
They observed a significant slowing
of diffusion just one minute after the injection. Thorne believes that their observations
help explain why electron microscopy images depict sizes between 10 and 20 nm. He
also said that the similar results help confirm that their method provides a good
estimate of the brain’s extracellular space.
The researchers would like to use the
integrative optical imaging technique to explore the diffusion of other —
perhaps larger — substances. They plan to investigate the diffusion
of materials that could be used in a drug delivery context as well.
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