Applied

Appl Magn Reson (2015) 46:623–632

DOI 10.1007/s00723-015-0670-7 Magnetic Resonance

Comparative Analysis by Magnetic Resonance Imaging

of Extracellular Space Diffusion and Interstitial Fluid
Flow in the Rat Striatum and Thalamus

Long Zuo1,2 • Kai Li3 • Hongbin Han1

Received: 19 October 2014 / Revised: 30 December 2014 / Published online: 26 March 2015
 Springer-Verlag Wien 2015

Abstract Drug delivery to the brain remains a challenge due to the blood–brain
barrier. Localized injection of drug therapies represents a promising alternative
once the diffusion characteristics of different brain regions have been evaluated.
Extracellular space diffusion and interstitial fluid flow of the striatum and thala-
mus in the rat brain were simultaneously compared using magnetic resonance
imaging and the tracer gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA).
The diffusion parameters, volume distribution, and half-life time were quantified.
While there was extensive diffusion of Gd-DTPA in the striatum, Gd-DTPA was
rapidly cleared and had a shorter half-life time in the thalamus. The increased
clearance rate and shorter half-life of the tracer in the thalamus were associated
with increased expression of Aquaporin-4. The tortuosity of the extracellular space
did not show a statistically significant difference between the two regions ex-
amined. Our research provides a new reference for brain interstitial drug delivery
to treat central nervous system diseases and a better understanding of the brain
microenvironment.

1 Introduction

The blood–brain barrier is a highly selective barrier that prevents the entry of most
drugs into the central nervous system (CNS) from the blood, which makes it difficult
to pharmacologically treat brain diseases. Currently, localized injection or

& Hongbin Han

hanhongbin@bjmu.edu.cn
1
Department of Radiology, Peking University Third Hospital, Beijing 100191, China
2
Beijing Key Lab of Magnetic Resonance Imaging Device and Technique, Beijing 100191,
China
3
Department of Radiology, Beijing Jishuitan Hospital, Beijing 100035, China

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convection-enhanced delivery represents the most straightforward approaches to

bypass the blood–brain barrier entirely, through targeted access to the extracellular
space (ECS) [1]. Because the characteristics of the ECS in different brain regions
are still poorly understood, localized injection of drugs into the brain has still not
been well explored [2]. The CNS contains extracellular fluid composed of
cerebrospinal fluid and interstitial fluid (ISF) [3]. Neurons within the ECS are
bathed by ISF, which contributes to the formation of a CNS microenvironment [4].
Recent studies have shown that both the cerebrospinal fluid and ISF play an
important role in the exchange and clearance of molecules in the ECS [5, 6]. In
diseases of the CNS such as Alzheimer disease and stroke, this clearance
mechanism is typically impaired [7]. Thus, understanding the physiological
characteristics of the ECS and ISF will greatly improve the treatment of CNS
diseases.
The studies mentioned above are limited, however, due to the use of in vivo
two-photon imaging of fluorescent tracers, where detection depth was limited to
the surface of the cortex. Technological improvements in magnetic resonance
imaging (MRI) have made it an emerging tool to assess ISF within the ECS to
better understand CNS-associated diseases [2]. Diffusion-weighted (DW)-MRI is
capable of measuring water molecule diffusion, but since water is present in both
the intra- and extracellular space simultaneously, interpretation of the studies is
quite challenging [8, 9]. The apparent diffusion coefficient measurement was
developed to analyze DW-MRI scans and provides a valuable means to analyze
diffusion in relation to specific brain structures, though the outcomes have been
more or less compromising [10, 11]. To overcome this obstacle, a new imaging
approach was developed, which utilizes gadolinium-diethylenetriaminepentaacetic
acid (Gd-DTPA) as a tracer to observe extracellular diffusion in vivo in rat brains
[12, 13]. This new technique allows quantitative measurements of ISF diffusion in
the ECS, which may provide a deeper understanding of ISF dynamics and
ultimately improve the efficiency of drug therapies provided to the brain interstitial
spaces.
Aquaporin-4 (AQP4) is the most abundant water channel found throughout brain
structures in contact with cerebral vascular compartments [14]. Emerging evidence
suggests that AQP4 plays a crucial role in brain water homeostasis [15] and is vital
for clearance of water from the brain into the vasculature [16]. Specifically,
astrocytic AQP4 has been associated with bulk flow to facilitate the perivascular
flow, which may serve as a clearance mechanism for some proteins such as cortical
beta amyloid [5]. Because astrocytic AQP4 localization differs significantly across
different brain structures [17–19], AQP4 may contribute to the different diffusion
and ISF flow parameters in respective brain regions.
In this study, we present the first data comparing the diffusion parameters and the
interstitial fluid properties within the ECS of the rat striatum and thalamus using
quantitative assessment of Gd-DTPA tracer injection. Moreover, we use our MRI
approach to demonstrate a correlation between AQP4 and ISF flow.

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2 Materials and Methods

2.1 Reagents and Experimental Animals

The study was conducted in accordance with the established Chinese guidelines for
the use of experimental animals, and the protocols were approved by the Ethics
Committee of Peking University Health Science Center (Approval No. LA2012-
016). Gd-DTPA (Magnevist; Bayer Schering Pharma AG, Berlin, Germany) was
diluted in double distilled water to a working concentration of 10 mmol/L. Mature,
age-matched Sprague–Dawley rats (n = 16) that weighed between 250 and 300 g
were used in this study. Rats were randomly divided into two groups: (1) striatum
injected (n = 8) and (2) thalamus injected (n = 8) [20].

2.2 MRI Pre-Scanning

An intraperitoneal injection cocktail of pentobarbital sodium, ethanol, chloral

hydrate, magnesium sulfate, and propylene glycol (3 mL/kg) was used to
anesthetize the rats. Anesthesia was subsequently maintained with additional
injections over the course of the experiment (approximately 0.7 mL/kg/h). Rats
were kept on a heating pad set at 38 ± 0.5 C to maintain body temperature and the
core temperature was monitored with a rectal thermometer at multiple time-points
throughout the procedure. To image the rat brain with MRI, a plastic animal
restraining device was used to fit the rats into a custom wrist coil. All rats were
scanned in a 3.0-Tesla MRI system (Magnetom Trio; Siemens Medical Solutions,
Erlangen, Germany) using a T1-weighted three-dimensional magnetization-pre-
pared rapid acquisition gradient echo (T1 3DMP-RAGE) sequence. Parameters for
the T1 3D MP-RAGE sequence were as follows: echo time (TE) = 3.7 ms,
repetition time (TR) = 1500 ms, flip angle (FA) = 12, inversion time
(TI) = 900 ms, slice thickness (SL) = 0.5 mm, field of view (FOV) = 267 mm,
voxel = 0.5 9 0.5 9 0.5 mm3 [21]. The MRI pre-scanning was performed to
acquire a reference image and establish puncture positions for the striatum and
thalamus. Volume of the whole brain was quantified using the 3DMP-RAGE images
from the MRI pre-scanning to calculate ratio of the maximal volume of distribution
(Vdmax) mentioned in Sect. 2.7.

2.3 Intraparenchymal Microinjection of Gd-DTPA

The skin overlying the calvaria was shaved and sterilized with iodated alcohol. A
scalp incision was made along the sagittal suture, between the interaural and
interocular areas. The skull bone was dissected away from membranes and muscle
attachments to expose the bregma suture. The rats were immobilized in a
stereotactic coordinate system (Lab Standard Stereotaxic-Single; Stoelting Co.,
Wood Dale, IL, USA). A microsyringe (Hamilton Bonaduz AG, Bonaduz,
Switzerland) was then stereotaxically positioned, such that 10 mmol/L of Gd-
DTPA solution could be manually infused into the striatum (needle placement

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measurements: anterior–posterior, ?1.0 mm; medial–lateral, -3.5 mm; dorsal–

ventral, -4.5 mm) [20] or the thalamus (needle placement measurements: anterior-
posterior, -2.0 mm; medial–lateral, -2.0 mm; dorsal–ventral, -5.5 mm) [20]
through a 2-mm burr hole at 0.2 ll/min for 10 min, followed by a 5 min pause to
avoid dorsal reflux along the needle track. Care was taken to minimize potential
damage inflicted by microsyringe penetration.

2.4 MRI and Post-Processing

Following intraparenchymal Gd-DTPA microinjection, rats were scanned every

30 min under the same conditions presented in Sect. 2.2. The Statistical Parametric
Mapping plug-in for MATLAB (MathWorks, Natick, MA, USA) was used to co-
register the MR images from the same rat pre-and post-injection. Pre-injection images
were used as masking for post-injection images to capture a region of interest enhanced
after Gd-DTPA injection. All voxels above a certain signal intensity threshold (i.e., two
standard deviations above the brain background noise) were outlined to calculate the
Gd-DTPA distribution volume at each post-injection time point.

2.5 Calculation of Diffusion Parameters

In a previous study from our lab, a direct linear relationship between signal
enhancement (DSI) and Gd-DTPA concentration was observed using a 3.0-T MRI
with a T1 3D MP-RAGE sequence [21]. This approach is easily carried out and is
equivalently accurate to the conventional method based on a 1/T1 measurement over
the same range at low concentrations [22–24]. The following MRI quantitative
assessments were made to calculate the diffusion parameters within the extracellular
space: free diffusion coefficient (D), effective diffusion coefficient (D*), tortuosity
(k), and clearance rate constant (k0 ) [12]. Based on the previous research from Li et al.
[9], we calculated the diffusion parameters. The free diffusion coefficient (D) of Gd-
DTPA was derived as 1.06 9 10-3 mm2/s using the least square-fitting method.

2.6 Volume Distribution and Half-life Time of Brain ISF Measurements

All voxels above the signal threshold (i.e., two standard deviations above the brain
background noise) in each region of interest were selected for calculating Gd-DTPA
distribution volume parameters at each time point post-injection. The whole brain
volume can be acquired from the three-dimensional scanning sequence in the Image
post-processing workstation software (Magnetom Trio). The ratio of maximal volume
of distribution (Vdmax) to the whole rat hemisphere’s volume can then be obtained.
A concentration–time curve was acquired from each voxel using sequential MRI
scans [21]; the tracer concentration topography on each slice was measured and the
integral of the total amount of Gd-DTPA tracer (Gdsum) was recorded for each time
point. The time-dependent Gdsum was assumed to be eliminated from the brain
based on a first-order kinetic model. The tracer clearance value was quantitatively
presented as a fitted curve of Gdsum (Gdsum = e-kt), where k is the clearance rate
and the tracer half-life (t) was calculated by the relationship thalf-life = ln(2/k).

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2.7 Immunohistochemical Analysis

Immunohistochemical analysis of the striatum and thalamus was performed as

described previously [25]. Five-micron sections were deparaffinized and incubated
in 3 % hydrogen peroxide. Sections were then incubated overnight at 4 C with a
primary antibody against AQP-4 (rabbit anti-rat; 1:300; Abcam, Cambridge, UK).
Sections were incubated with secondary antibody for 30 min at 37 C. The color
reaction was developed using 3,30 -Diaminobenzidine, and nuclei were counter-
stained with hematoxylin and eosin (HE).

2.8 Western Blot Analysis

Aquaporin-4 levels in the rat brain were detected by western blotting, as previously
described [26]. Briefly, 50 lg of each sample was loaded onto a 12 % Tris
polyacrylamide gel and subsequently transferred to a polyvinylidene difluoride
membrane (Bio-Rad, Hercules, CA, USA). Membranes were blocked for 1 h with
5 % nonfat milk and incubated overnight at 4 C with a primary antibody against
AQP-4 (rabbit anti-rat; 1:1000; Abcam) or a monoclonal antibody against GADPH
(1:10,000; Abcam) in TBST (10 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 0.05 %
Tween 20) containing 3 % nonfat milk. Membranes were then incubated for 1 h at
room temperature with goat anti-rabbit secondary antibody. Proteins were detected
by chemiluminescence and analyzed with ImageJ software (National Institutes of
Health, Bethesda, MD, USA).

2.9 Statistical Analysis

Statistical analysis was conducted using SPSS statistical software, version 19.0
(IBM SPSS Statistics for Windows, Armonk, New York, USA). All values are
expressed as the mean ± standard deviation (SD). To compare the free diffusion
coefficient (D), effective diffusion coefficient (D*), tortuosity (k) and clearance rate
constant (k0 ), maximal volume distribution (Vdmax) and half-life time (thalf-life)
between the striatum and thalamus, independent sample t-tests were used. Paired t-
tests were used to compare differences between western blotting analyses and
immunofluorescence parameters in the striatum and thalamus. A p value of \0.05
was set as the threshold for statistical significance.

3 Results

3.1 Comparison of the Gd-DTPA Diffusion Parameters Between the Rat

Striatum and Thalamus

After the Gd-DTPA tracer was microinjected into the ECS, the signal intensity of
the striatum (Fig. 1a) and thalamus (Fig. 1b) increased. The hyperintensity then
attenuated after the tracer was distributed and cleared over time (Fig. 1a, b). When
the striatum and thalamus were compared, we observed no significant difference in

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Fig. 1 Diffusion parameters of Gd-DTPA in the striatum and thalamus. a Coronal views of the striatum
over time before and after injection of Gd-DTPA. b Coronal views of the thalamus over time, pre- and
post-injection of Gd-DTPA. c Quantitative analyses of diffusion parameters (D*, k, k0 ) in the striatum and
thalamus. Data are presented as the mean ± SD (n = 8). **p \ 0.01

the effective diffusion coefficient (D*) [(3.33 ± 0.69) 9 10-4 vs. (3.37 ± 0.45) 9
10-4 mm2/s; t = 0.12; p = 0.91]. The lack of difference in D* led to no significant
difference in the tortuosity (k = (D/D*)1/2) [(1.81 ± 0.22) vs. (1.29 ± 0.22);
t = 0.13; p = 0.90] (Fig. 2c). In contrast, however, we observed a significant
difference in the clearance rate constant (k0 ), which was lower in the striatum
[(0.64 ± 0.11) 9 10-4 mm2/s] than in the thalamus [(1.55 ± 3.94) 9 10-4 mm2/s;
t = 5.416; p \ 0.001].

3.2 Comparison of the Gd-DTPA Distribution and Half-life Time Between

the Rat Striatum and Thalamus

When the striatum was injected with Gd-DTPA, the tracer predominantly diffused
into the middle cerebral artery (MCA) region 2 h post-injection and maintained a high
signal intensity there for 2–4 h (Fig. 2a, c). When the thalamus was injected, the Gd-
DTPA tracer was largely retained in the thalamus area and the signal intensity
decreased rapidly beginning 30 min post-injection through 3 h post-injection
(Fig. 2b, c). The maximal distribution volume of the Gd-DTPA tracer in the striatum
(210.7 ± 34.5 lL) was larger than that of the thalamus (170.45 ± 16.68 lL)
(Fig. 2c). Accordingly, the volume ratio of the striatum was larger than that of the
thalamus [(9.61 ± 1.36 %) vs. (2.30 ± 0.62 %); t = 11.95; p \ 0.001] (Fig. 2d).
The half-life time of the thalamus (0.81 ± 0.03 h) was significantly shorter than in
the striatum (1.40 ± 0.12 h) (t = 11.16; p \ 0.001) (Fig. 2e).

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Fig. 2 Spatiotemporal distribution of Gd-DTPA injection into the striatum and thalamus. a ISF drainage
of the striatum was obtained by the computer-aided pseudocolor enhancement method and is shown in
sagittal (the first row), axial (the second row) and horizontal (the third row) views. b ISF drainage from
the thalamus. c Changes in the Gd-DTPA distribution volume in the striatum and thalamus over time.
d The Vdmax of the striatum and thalamus. e The half-life time of the GD-DTPA in the striatum and the
thalamus. Data are presented as the mean ± SD (n = 8). **p \ 0.01

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Fig. 3 AQP4 expression in the striatum and thalamus. a Representative HE-stained paraffin sections of
the striatum (magnification: 9200). Patch compartments are indicated (arrows). b Representative sections
of AQP4 immunohistochemistry in paraffin sections of the striatum (magnification: 9200).
c Representative HE-stained paraffin sections of the thalamus (magnification: 9200). d Representative
AQP4 immunohistochemistry in thalamus paraffin sections (magnification: 9200). e Representative
western blot for AQP4 in the striatum and thalamus. AQP4 levels were higher in the thalamus than the
striatum. Data are presented as the mean ± SD (n = 6). **p \ 0.01

3.3 Histological Characteristics and AQP4 Expression in the Rat Striatum

and Thalamus of Rat Brains

To better understand the differences in the clearance rate constant (k0 ) and half-life
time observed between the striatum and thalamus, HE histology, immunohisto-
chemical analysis and immunoblot analysis were performed. Comparison of HE
histological sections of the striatum (Fig. 3a) and thalamus (Fig. 3c) revealed patch
(striosome) compartments in the striatum, as was previously reported [27, 28].
Immunohistochemistry for AQP4 in the striatum and thalamus revealed more
extensive expression ofAQP4 in the thalamus (Fig. 3d). In contrast, AQP4
expression in the striatum was limited to areas surrounding vessels (Fig. 3b). In
accordance with the immunohistochemical results, immunoblots for AQP4 revealed
significantly higher levels in the thalamus in comparison to the striatum (p \ 0.01)
(Fig. 3e).

4 Discussion

In the present study, the extracellular contrast agent Gd-DTPA [29] was employed
to visualize diffusion into the ECS adjacent to the striatum and the thalamus [12].
As well known, the magnetic resonance signal intensity is only proportional to the
number of nuclear spins in the pixel which is not changed by introducing Gd-DTPA.
Gd-DTPA is effective for increasing T1 relaxation rate (1/T1) and commonly used

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as T1 contrast agents, generating a positive image contrast which represents high

signal intensity on MRI. In the current study, the thalamus showed higher clearance
rate and higher signal intensity following tracer injection and these features
correlated with an increased expression of AQP4, as compared to the levels
measured in the striatum.
Differences in the striatum and thalamus ISF characteristics were observed in the
clearance rate constant (k0 ), half-life time (thalf-life), and volume distribution (Vdmax).
The diffusion patterns of Gd-DTPA were significantly different between the
striatum and thalamus. While the striatum showed extensive diffusion, the thalamus
showed a rapid clearance of the tracer. Injection into the striatum resulted in tracer
accumulation predominantly into the MCA region (Fig. 2a) [30]. Consequently, the
greatest Vdmax was observed in the striatum (Fig. 2d) due to the more extensive
distribution from the point of injection to the ipsilateral cortices. The Gd-DTPA
distribution in the thalamus displayed a greater clearance rate constant, due to the
shorter half-life time. The thalf-life (derived from curve fitting of the MRI signal
intensity attenuation with time) in the thalamus is less than that of striatum, which is
consistent with the k0 (calculated according to the diffusion equation [12]) in the
same two areas. The different characteristics of the two regions examined suggest
that different brain regions adjust to substrate delivery based on the endogenous
properties of each region.
This research revealed a different clearance rate constant and half-life time in the
thalamus versus the striatum, which was correlated with differential expression of
AQP4. AQP4, the predominant aquaporin expressed in the brain [31], is expressed
in glial cells and absent in neurons [18]. AQP4 is a plasma membrane protein
concentrated in astrocyte membrane domains that oppose vessels and pia [32] to
facilitate the exchange of water between ISF and extracerebral liquids such as blood
and cerebrospinal fluid [33]. Recent studies have shown that AQP4 plays a critical
role in the clearance of potentially harmful substances from the CNS [5, 14]. Our
data are consistent with the previous research and show that elevated AQP4
expression in the thalamus is associated with a faster clearance constant (Fig. 1c)
and a shorter half-life time (Fig. 2e) in comparison to the striatum (Fig. 3e). Future
studies should explore the cellular and molecular mechanisms underlying ECS
diffusion and ISF flow.
The present research demonstrates the physiological parameters of the ECS in the
striatum and thalamus and has potential clinical applications. Using the methods
established here, the delivery of therapeutic agents interstitially to brain can be
performed to provide a reference index for drug diffusion and clearance within brain
ECS, factors that are crucial for dosage regimen design. Because the blood–brain
barrier remains an obstacle in effectively treating the CNS with drug therapies [34],
local brain delivery by direct injection or infusion of drugs into the brain represents
a promising method for targeted drug delivery strategies [35–37].

Acknowledgments This work was supported by the National Natural Science Foundation of China (No.
81171080, No. 61450004), National key developmental program for scientific instrument and equipment
(Grant No. 2011YQ030114), and the Key Project in the National Science and Technology Pillar Program
during the Twelve Five-year Plan Period of China (2012BAI15B009).

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