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IMAGING THE PERIVASCULAR SPACE 67

vessels may be critically important in the removal and restricted to ~250µm below the cortical surface in
transport of macromolecules from CSF to peripheral recent studies (36,37,45,79). This depth does not
lymph nodes (49). As such, the meningeal lymphatic permit the measurement of fluid influx and efflux
vessels are a key target of future research. routes along subcortical brain regions in vivo, and
thus requires the use of fluorescence microscopy for
IMAGING FLUID PATHWAYS IN ANIMALS ex vivo analysis. While progress has been made to
increase the imaging depth of multiphoton micros-
Fluorescence Microscopy copy (26,32,44), present technology still limits the
Fluorescence microscopy is the most common examination of CSF and ISF movement in real time
method for imaging fluid movement in brain slices to cortical areas.
and is subject to a number of excellent reviews Bedussi and colleagues have recently used serial
(48,75,77). Standard procedures examining the brain slices to create computer-generated 3D models
movement of CSF/ISF use fluorescence microscopy of the mouse vasculature (8). This innovative imaging
to quantify light emitted from brain slices stained technique has allowed the authors to visualize the
with dyes containing fluorophores. Fluorescent dyes distribution of tracers along brain-wide networks of
are commonly administered to CSF by injection into perivascular flow. Their findings, specifically regard-
the ventricles (intraventricular), cisterns (intracis- ing ISF efflux, show that intracerebral tracers moved
ternal), or spinal cord (intrathecal), while ISF is by bulk flow toward the ventricles (8) rather than
usually accompanied by injection directly into the perivenous pathways as proposed by the glymphatic
brain (intracerebral). Both intraventricular and intra- hypothesis (36). This comparison helps frame the
cisternal injections are standard practice for studying importance of utilizing more advanced imaging
the distribution of tracers throughout brain-wide techniques to measure perivascular processes and
networks of perivascular flow. Intracerebral injec- demonstrates why further procedural and techni-
tions are utilized when the activity and movement cal development is necessary to clarify inconsistent
of solutes and waste of the ISF must be examined results.
directly. While intrathecal injections are also used
to examine global fluid movement, this application Contrast-Enhanced MRI
is perhaps transferable into clinical settings and is MRI allows for the noninvasive visualization
further explained in the next section (8). of healthy or pathological brain tissue, which has
Multiphoton microscopy is a powerful imaging been widely used for both clinical research and
technique capable of detailing the three-dimensional diagnostic purposes. Recent animal studies using
morphology of biological structures tagged with MRI have utilized lumbar intrathecal injections of
fluorescent stains (14,70). One major advantage of gadolinium-based contrasts and were successfully
this imaging technique is the in vivo application to able to examine CSF-ISF exchange in vivo (20,34,81).
examine perivascular pathways in animals. Iliff et al. Additionally, the dispersion rates of contrasts with
utilized in vivo two-photon microscopy to visualize different molecular weights were analyzed. Regardless
the real-time movement of tracers along perivascu- of molecular weight, contrasts were transported at
lar spaces (36). Their findings revealed that tracers similar rates, indicating that bulk flow, aside from
injected into the CSF of the cisterna magna traveled basic diffusion, can be examined using contrast
along the exterior of arteries projecting into the brain enhanced MRI (34). These results are consistent with
from the SAS (36). Others have also used multiphoton the basic principles of the glymphatic hypothesis
microscopy to examine ISF clearance in vivo, show- and show that bulk flow, which is the main mecha-
ing that tracers injected intracerebrally accumulate nism driving interstitial clearance, can be examined
around arteries and capillaries (4). via contrast-enhanced MRI (34,81). Unfortunately,
Despite the strengths, multiphoton microscopy is contrast-enhanced MRI cannot yet identify spe-
limited to cortical brain areas with imaging depths cific biological variations that alter glymphatic flow.