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 space. 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 tissue. 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 space. 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 said. 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,” Thorne said. 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.