NANO

LETTERS

Nanowire Biocompatibility in the Brain Looking for a Needle in a 3D Stack

2009
Vol. 9, No. 12
4184-4190

Cecilia Eriksson Linsmeier,*,,| Christelle N. Prinz,*,,,| Lina M. E. Pettersson,

Philippe Caroff,, Lars Samuelson, Jens Schouenborg, Lars Montelius,
and Nils Danielsen
Neuronano Research Center, BMC F10, Lund UniVersity, SE-221 84 Lund, Sweden,
and DiVision of Solid State Physics, Lund UniVersity, SE-221 00 Lund, Sweden
Received July 27, 2009; Revised Manuscript Received September 25, 2009

ABSTRACT
We investigated the brain-tissue response to nanowire implantations in the rat striatum after 1, 6, and 12 weeks using immunohistochemistry.
The nanowires could be visualized in the scar by confocal microscopy (through the scattered laser light). For the nanowire-implanted animals,
there is a significant astrocyte response at week 1 compared to controls. The nanowires are phagocytized by ED1 positive microglia, and
some of them are degraded and/or transported away from the brain.

Neural interfaces are expected to play an important role for

diagnosis and therapy of clinical conditions such as pain
control, facilitation of motor control after stroke, trauma, or
neuro-degenerative disorders.1,2 Such interfaces need to be
chronically implanted into the central nervous system (CNS)
to stimulate and record nerve signals while being biocompatible. It has been shown that nanostructured surfaces can
improve electrical properties of electrodes and elicit reduced
tissue responses.3 While there are a few in vivo studies of
carbon nanotube biocompatibility in the literature,4-6 there
are no in vivo studies of nanowire biocompatibility. We have
shown7-9 that neurons can thrive on gallium phosphide (GaP)
nanowire substrates, even when penetrated by the nanowires.
This indicates that the GaP nanowires are biocompatible in
the in vitro situation, and it opens up the possibility of
recording from individual neurons since the cell-electrode
distance can be made very small. It is now possible to record
and stimulate single neurons using nanowire transistor
arrays.10 In our lab, we are planning to use vertical nanowires
on the surface of a microelectrode for brain implantation.
Before using such electrodes, it is necessary to investigate
what would happen if the nanowires detach from the
electrode after implantation. This could be considered as a
worst-case scenario with high aspect ratio particles deposited
* Corresponding author. (C.E.L.) Phone: +46 46 222 4107; fax: +46
46 222 7756; electronic mail address: Cecilia.Eriksson_Linsmeier@med.lu.se.
(C.N.P.) Phone: +46 46 222 4796; fax:+46 46 222 3637; electronic mail
address: christelle.prinz@ftf.lth.se.

Neuronano Research Center, BMC F10, Lund University.

Division of Solid State Physics, Lund University.

Present address: Institut dElectronique de Microelectronique et de

Nanotechnologie, UMR CNRS 8520, Avenue Poincare, BP 60069, F-59652
Villeneuve dAscq, France.
|
These authors contributed equally to this work.
10.1021/nl902413x CCC: $40.75
Published on Web 10/21/2009

2009 American Chemical Society

in the tissue. To this end, we investigated the tissue response

after nanowire injection into the brain. Our approach is to
address the biocompatibility of the nanowire as a nanorod
and to eliminate possible concerns due to the chemical nature
of the nanowires by coating them with a chemically
biocompatible material. In order to do so, we have used GaP
nanowires covered with SiOx. It is known that SiOx surfaces
have biocompatible properties and that only their size and
shape are a potential hazard for the body.11,12 GaP, on the
other hand is known to partially dissolve and accumulate in
the kidney and liver when implanted in the rat soft tissue
and therefore cannot be considered as a chemically biocompatible material.13 The nanowires were suspended in a
physiological solution and implanted in the rat striatum. The
tissue response was investigated 1, 6, and 12 weeks after
the implantation with respect to quantification of crucial
components: microglia (detected by the membrane-bound
ED1-antigen), astrocytes (detected by their production of glial
fibrillary acidic protein, GFAP), neurons (detected by
antibodies directed against neuronal nuclei, NeuN), and the
total number of cell nuclei using a specific staining named
DAPI and by localizing the nanowires in the injected area.
The microglia detect and engulf any infectious substance as
well as dead cell debris. As part of the inflammatory
response, they secrete various substances to destroy harmful
agents. However, these substances are also toxic to the
remaining neurons, and extensive damage to the brain may
results from the inflammatory reaction itself.14 Astrocytes
are activated upon brain injury, and one step in this activation
is an increased synthesis of GFAP. The role of the astrocytes
is not fully understood; they have opposing effects after a
traumatic brain injury.15,16 They are involved in brain-tissue

repair; they also form a protective scar around the injury,

limiting the spread of the inflammatory response to the
injured area. Astrocytes have also been shown to inhibit
axonal regeneration. On the basis of these facts, quantification
of the microglia, astrocytes, cell nuclei, and NeuN should
provide a good assessment of the tissue response to the
nanowire implantation.
The nanowires were grown in a standard commercial metal
organic vapor phase epitaxy (MOVPE) reactor at a working
pressure of 104 Pa. Prior to insertion in the reactor,
GaP(111)B substrates were decorated with 80 nm diameter
pure single crystalline gold seed particles at a surface density
of 1.0 m-2, using a dedicated aerosol setup.17 The samples
were then transferred into the growth chamber where they
were first annealed at 600 C for 10 min under a constant
partial pressure of phosphine (PH3) in a hydrogen carrier
gas flow of 13 L min-1 to deoxidize the epi-ready samples
and allow the eutectic alloy between gold seed particles and
gallium from the substrate to form.18 After the annealing step,
the growth temperature was ramped down to a nanowire
growth temperature of 470 C. GaP nanowires were grown
for 4 min, using trimethylgallium (TMGa) and PH3 as group
III and group V material precursors, respectively. Molar
fractions in the range of 1.2 10-5 and 8.5 10-3,
respectively, for TMGa and PH3 were typically used to grow
the nanowires, giving a nominal V/III ratio of 708. Growth
was terminated by stopping the TMGa flow, and temperature
was ramped down to room temperature under a protective
PH3/H2 flow identical to the one used during growth, in order
to avoid phosphorus desorption, which would be detrimental
for the nanowire quality. The resulting single crystalline GaP
nanowires had a diameter of 80 nm and a length of 2 m,
with low tapering and high vertical yield.
The nanowire substrates where coated with 60 nm of
sputtered SiOx (corresponding to 20 nm on the nanowire
walls) (Orion 5 sputterer, AJA International). All substrates
were cleaned in an oxygen plasma chamber (Plasma Preen)
to make the nanowires sterile and hydrophilic. All tubes,
tweezers, liquids, and pipette tips used from this point were
sterile. The nanowire substrates were individually dipped in
an Eppendorf tube containing Hanks balanced salt solution
(HBSS). The amount of HBSS was chosen so that the final
concentration of nanowires was 70 000/L. Each Eppendorf
tube was then sealed and immersed in an ultrasound bath
for 2 min to break off the nanowires from the substrate. The
GaP wafer piece was removed from the tube, air-dried, and
the absence of nanowires from the surface was checked using
a scanning electron microscope (SEM). As an additional
precaution, a drop of the nanowire solution was deposited
on a microscope slide, and the presence of nanowires was
verified using confocal microscopy. The nanowires are
clearly visible in confocal microscopy through the scattered
laser light. The nanowire solution was kept at 4 C until the
implantation surgery.
We used a total of 25 adult female Sprague-Dawley rats
(Taconic, Denmark). This study was approved by the Malmo/
Lund animal ethics committee on animal experiments. The
surgical procedures were made under strict sterile conditions.
Nano Lett., Vol. 9, No. 12, 2009

Figure 1. Fluorescence microscopy image of the scar one week

after nanowire implantation. The ED1-positive cells (green), the
GFAP-positive cells (red), and the cell nuclei (blue) were quantified
by measuring the relative (%) area for each individual stain within
the 100 m 800 m box (in gray) around the scar. Scale bar
200 m.

The rats had a weight of approximately 220 g at the

beginning of the experiment and followed a normal weight
curve after the implantation. All the animals were housed
under a 12 h light/dark cycle with free access to water and
food. Before the implantation surgery, the rats were deeply
anesthetized by intraperitoneal (i.p.) injections of a mixture
of fentanyl (50 mg/mL) and Domitor vet 1 mg/mL (medetomidin hydrochloride) as previously described.13 They were
then prepared for surgery by shaving the head and placed in
a Kopf stereotaxic frame set under a stereomicroscope
(Leica). The surgical area of the scalp was disinfected using
70% ethanol solution, and a 3 cm midline incision was made
to expose the skull. The tissue attached to the skull was
carefully removed and blood was cleansed away. Bilateral
stereotaxic injections were made from glass microcapillaries
attached to a 2 L Hamilton syringe. The nanowire suspension was injected bilaterally into the striatum (coordinates
(mm): A ) 1.0; L ) (2.5; V ) (a) -5 (1 L), (b) -4 (1
L)), in total 2 L/side over a total time period of 4 min.
Finally, for all animals implanted, the skin was closed using
surgical clips (Michell 7.5 1.75). For the 1 week (6 week,
12 week) implantation period, 4 (4, 5) rats were implanted
with a SiOx-coated GaP nanowire suspension, and, as a
control, 4 (4, 4) rats were implanted with HBSS only. The
surgeries for the three different groups (1, 6, and 12 weeks)
took place in three different sessions. After each injection
series, a drop of the suspension was poured from the same
syringe used for the surgery onto a miscoscope slide, and
the presence of suspended nanowire was checked using
confocal microscopy. After surgery, the animals received
subcutaneous injections of 1 mL/kg body weight of a mixture
of Antisedan vet 5 mg/mL (atipamezole hydrochloride) and
sterile water. This injection serves as an antidote to the
anesthesia. At the same time the animals received analgesia
subcutaneously. After 1, 6, and 12 weeks, respectively, the
animals were anaesthetized with an overdose of pentobarbital
(i.p.) and transcardially perfused with 150-200 mL of ice4185

and sent to ALS Analytica AB (Lule, Sweden) for analyses

of the gallium content in the different tissues. No gallium
was detectable in the organs or blood samples.
In order to detect the important cell components, a number
of different immunohistochemical stainings were performed.
The first series of brain sections was stained as follows: The
sections were rinsed in phosphate buffered saline (PBS, 0.02
M, pH 7) and preincubated in a blocking solution consisting
of 5% normal goat serum and 0.25% Triton X-100 in 0.02
M PBS for 1 h at room temperature. The sections were then
incubated with the primary antibodies mouse-anti-ED1
(microglia), 1:200 (CD68; AbD Serotec) and a rabbit-antiGFAP (astrocytes), 1:5000 (DAKO) overnight at room
temperature. After repeated rinsing in PBS, they were further
incubated with 4,6-diamidino-2-phenylindole (staining all
cell nuclei) 1:1000 (DAPI, Invitrogen), antimouse-Alexa488 and antirabbit-Alexa-594 conjugated secondary antibodies 1:500 (Invitrogen) for 2 h in the dark, at room
temperature. All primary and secondary antibodies were
diluted in the blocking solution mentioned above. The
sections were then rinsed in PBS and coverslipped, with
polyvinyl alcohol mounting medium with 1,4-diazabicyclo[2.2.2]octane (DABCO; Fluka analytical). For all the different antibody protocols, controls by omission of primary
antibodies were negative.
All histological fluorescence images were obtained using
a DS-2Mv Digital camera (Nikon), mounted on a Nikon
Eclipse 80i microscope with a 10 objective (Nikon). The
images were acquired and analyzed using the NIS-Elements
BR software (NIS-Elements).

Figure 2. Quantification of ED1-positive cells, GFAP-positive cells,

cell nuclei, and NeuN at weeks 1, 6, and 12 for the nanowireimplanted animals and the controls. The box (white for controls,
gray for nanowires) corresponds to the 25, 50 and 75 percentile,
and the whiskers show the minimum and maximum values. The
horizontal lines indicate statistical differences.

cold 4% paraformaldehyde (PF) in 0.1 M phosphate buffer

(PB). All the brains were carefully removed and postfixed
at 4 C overnight in fixative (4% PF) and rinsed overnight
in 0.1 M PB containing 25% sucrose for cryopreservation.
The brains were sectioned in 10 m thick slices in the coronal
plane in a cryostate. The brain slices were put on microscope
slides (8 per slide). Every other consecutive section was put
on a second microscope slide, allowing us to have two almost
identical series of brain slices for making two series of
immunostainings. For the 12-week implantation group,
samples of blood, kidneys, and liver were taken, snap frozen,
4186

The slices were screened to detect the scar corresponding

to the -4 mm and -5 mm injections, where most of the
ED1- and GFAP-positive cells are located. A photograph of
the fluorescence of the ED1-positive cells (green), GFAPpositive cells (red), and cell nuclei (blue) was taken at the
maximum of the scar for each injection site (four photographs
per animal). The adjacent brain slices belonging to the second
series were then stained according to the protocol described
above without the ED1 staining but with NeuN staining
instead: the sections were incubated with mouse anti-NeuN
(Millipore) 1:100 in the blocking solution overnight at room
temperature and were then incubated with antimouse Alexa
Fluor 488 according to the protocol described above. A
photograph of the NeuN fluorescence was taken for each
injection site. The different cell-type quantifications were
made by defining a 100 m by 800 m rectangle around the
scar for each of the photographed sections and then measuring the proportion of immunoreactive area in the total
screened area for each marker. The threshold for the ED1positive cells, GFAP-positive cells, NeuN, and cell nuclei
(DAPI) was chosen to be 6, 4.5, 2.5 and 4 times above the
background, respectively. Figure 1 shows a fluorescence
microscopy image of the scar week 1 after the implantation
of nanowires. The quantification box is shown in gray. For
evaluation of effects over time for HBSS-injected and
nanowire-implanted animals, the Kruskal-Wallis test (K.W.)
was used with the Dunns test as the posthoc test.
Nano Lett., Vol. 9, No. 12, 2009

Figure 3. Laser scanning confocal microscopy 3D stacks of the scar for nanowires at 1, 6, and 12 weeks. From top to bottom: ED1-positive
cells (green), GFAP-positive cells (red), cell nuclei (blue), nanowires (white), and a composite of all elements. Scale grid unit 14.3 m. The
insets correspond to a 24.5 31.5 0.8 m3 slice taken from the z-stack and illustrate the nanowire distribution in the scar at weeks 1,
6, and 12. Note the single nanowires at week 1.

For comparison between HBSS-injected and nanowireimplanted animals at specific time points the Mann-Whitney
U test (M.W.) was used. The statistical program used was
GraphPad Prism; p < 0.05 was considered significant. Figure
2 shows the area fraction (%) of the ED1 and GFAP-positive
cells, cell nuclei (DAPI), and NeuN for the nanowires and
the controls at weeks 1, 6, and 12. In the nanowire-implanted
animals, there was a significant decrease of ED1-positive
cells between weeks 1 and 6 and between weeks 1 and 12,
indicating that the initial evoked inflammatory response
declined over time. The astrocyte response (GFAP) at week
1 was largely increased for nanowire-implanted animals as
compared to controls (M.W.). For the nanowire-implanted
animals, this response did decline over time, although it did
not reach statistical difference. For the control animals, there
was a small increase in the response between week 1 and
12 (K.W.). At week 1, the main difference found between
the two groups was the increased GFAP response for
nanowire-implanted animals as compared to controls.
For cell nuclei, control animals displayed an initial
response which declined over time, indicated by significantly
smaller numbers at week 6 as compared to week 1 and
between week 12 and week 1 (K.W.) A similar pattern could
be observed for the nanowire-implanted animals. The
significant decrease occurred between weeks 1 and 6, as well
as between weeks 1 and 12 (K.W.). However, at week 12,
there were more cell nuclei in the scar for nanowires than
Nano Lett., Vol. 9, No. 12, 2009

for controls. Finally, no significant differences were found

between the two populations with respect to the proportion
of NeuN at weeks 1, 6, and 12. The normal values (measured
far away from the scar) are given in the Supporting
Information and compared to the control values. These data
mainly give information on the effect of the needle hole in
the brain and are not very relevant when the specific effect
of the nanowire implantation is investigated.
The brain slices used for cell quantification were subsequently analyzed using confocal microscopy for nanowire
localization. Three-dimensional (3D)-stack images of the scar
were made using a laser scanning confocal microscope (Zeiss
LSM 510) with a 63 oil-immersion objective (N.A. 1.4)
and the Volocity 4.0.1 imaging software. The images were
acquired in multitrack mode with a z-slice of 0.8 m (one
Airy Unit for the Argon laser) and a stack step size of 0.4
m. The nanowires (as well as any inorganic matter) are
visible in the laser-reflection mode since they scatter the laser
light much more than the brain tissue. Figure 3 shows 3D
stacks of the scar 1, 6, and 12 weeks after the nanowire
injection. The color labeling is the same as before (Green:
ED1-positive cells/ Red: GFAP positive cells/ Blue: Cell
Nuclei). The nanowires are shown in white. The insets show
a 24.5 m 31.5 m single slice (0.8 m thick) taken from
the same z-stack. The insets have been chosen with respect
to the nanowire distribution; they do not necessarily reflect
the cell quantification results shown in Figure 2.
4187

Figure 4. Laser scanning confocal microscopy 3D stacks of the scar for HBSS buffer injection (control) at 1, 6, and 12 weeks. The
ED1-positive cells are green, the GFAP-positive cells are red, and the cell nuclei are blue. Scale grid unit 14.3 m.

Figure 5. z-stack close-up of nanowires clusters at week 1 (a), week 6 (b), and week 12 (c) (green: ED1; red: GFAP; blue: cell nuclei;
white: nanowires). The clusters are inside ED1-positive cells at weeks 1, 6, and 12. Scale grid units: 2.4 m (a), 2 m (b), and 2.4 m (c).

At one week, the nanowires were spread out along the

scar. Most of the nanowires were isolated, and only a few
nanowire clusters (three on the 3D stack image of Figure 3)
were formed in the ED1-positive cells. At week 6, the cluster
size and number increased compared to week 1, and fewer
single nanowires could be found. At 12 weeks, most of the
nanowires have been phagocytized by the ED1-positive cells.
No signs of frustrated phagocytosis were seen in the
microglia, and the fact that the proportion of ED1-positive
cells did not increase at week 6 and 12 suggests that there
was no subacute or chronic toxicity. Figure 4 shows 3Dstacks of the control animals at week 1, 6, and 12. Note the
absence of nanowires. A little bit of inorganic matter could
be seen in the week 1 and 6 images, which probably
4188

corresponds to some glass debris from the capillary used for

the injection.
Although the astrocytes were strongly involved in the
tissue response to nanowire implantation at week 1, the
engulfing of most of the nanowires was probably done by
the migroglia at all time points, as shown in the z-stacks in
Figure 5. It is possible that the astocytes were activated
during the first week of the injection to create a barrier to
prevent the nanowires from diffusing further into the brain
until the microglia engulfed them. More experiments would
be needed to confirm this hypothesis. For example, one could
redo the experiments with GFAP-TK transgenic mice, for
which the reactive astrocytes can be selectively ablated.19,20
A wider spread of the nanowires for the transgenic mice
Nano Lett., Vol. 9, No. 12, 2009

at week 12. For that particular animal, no intact nanowires

were found at all, suggesting that they have all been reduced
to dust. The nanowire degradation can possibly be explained
by the fact that ED1-positive cells secrete hydrogen peroxide.
Hydrogen peroxide can etch the inner GaP core of the
nanowires (data not shown), leaving a fragile SiOx shell that
can be easily broken by the cells.14
In conclusion, we have investigated the biocompatibility
of nanowires in the rat brain. Nanowire implantation elicited
a rather large astrocyte reaction during the first week. This
activation declined over time. Nanowire-implantation also
induced a microglial (ED1) response, which declined over
time. There was no significant difference in the neuronal
fraction for the nanowire-implanted animals compared to
controls at all time points. ED1-positive cells engulfed the
nanowires, a phenomenon that was amplified at weeks 6 and
12. Some of our data suggests that some nanowires were
able to pass the blood-brain barrier and leave the brain. We
also show evidence of nanowire partial degradation inside
the ED1-positive cells at weeks 6 and 12. The rats followed
a normal weight curve during the experiment. No evidence
of frustrated phagocytosis was observed. We have no
evidence of subacute or chronic nanowire toxicity. Nevertheless, a greater GFAP response at week 1 and an increase in
cell nuclei at week 12 were detected for the nanowireimplanted animals as compared to controls, indicating that
the nanowires have short- and long-term effects that require
precaution and further investigation.

Figure 6. Possible mechanisms of removal/degradation of nanowires. (a) 3D-stack of nanowires inside two ED1-positive cells in a
blood capillary at week 1. The capillary was located 100 m away
from the scar. Scale grid unit: 4.8 m. (b) 3D-stack of degraded
nanowires inside an ED1-positive cell at week 6 (only the degraded
nanowires are shown). Scale grid unit: 2 m. (c) 3D-stack of
degraded nanowires inside five ED1-positive cells at week 12 (only
the degraded nanowires are shown). Scale grid unit: 14.3 m.

compared to wild-type mice would then confirm the role of

the astrocytes as barrier for the nanowires.
In some 3D stacks, we could see that the nanowires were
being partially degraded and/or transported away from the
brain. Figure 6a shows a 3D-stack of nanowires at week 1
being removed from the brain. Nanowires engulfed by ED1positive cells were found inside a blood capillary. The
capillary was located 100 m away from the scar, which
suggests that, either the nanowires have diffused to the
capillary site or they have been transported from the scar to
the capillary by the microglia. While most of the microglial
cells at week 6 and 12 contained rod-like shape nanowires,
some of them contained degraded nanowires. Figure 6b
shows a 3D-stack of a degraded nanowire cluster inside an
ED1-positive cell (only the laser reflection mode is shown
here). The debris no longer appeared rod-shaped, as was the
case for the intact nanowires. Figure 6c shows a 3D-stack
of further degraded nanowires inside five ED1-positive cells
Nano Lett., Vol. 9, No. 12, 2009

Acknowledgment. We thank Jonas Thelin, Konstantin

Vogel, Mark Frick, Lina Gallentoft, Linda Faxius, Dmitry
Suyatin, and Suzanne Rosander for their help. This work
was initiated and done within the Neuronano Research Center
in collaboration with the Nanometer Structure Consortium,
both at Lund University. The project was funded mainly by
the Swedish Research Council (Linne Grant Project Number
600012701) and the Knut and Alice Wallenberg Foundation
(Project Number KAW 2004-0119).
Supporting Information Available: Normal values (quantification far way from the scar) of GFAP-positive cells, cell
nuclei, and NeuN are shown and compared to the control
values. This material is available free of charge via the
Internet at http://pubs.acs.org.
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NL902413X

Nano Lett., Vol. 9, No. 12, 2009