Intraspinal delivery of polyethylene glycol coated gold nanoparticles promotes
functional recovery after spinal cord injury
F. Papastefanaki1, I. Jakovcevski2,3#, N. Poulia1#, Nevena Djogo2, F. Schulz4, T.
Martinovic5, D. Ciric5, G. Loers2, T.Vossmeyer4, H. Weller4,6, M. Schachner7*, R.
Matsas1*
1. Laboratory of Cellular and Molecular Neurobiology, Hellenic Pasteur Institute, 127
Vassilissis Sofias Avenue, 11521 Athens, Greece
2. Zentrum für Molekulare Neurobiologie, Universitätsklinikum Hamburg-Eppendorf,
Universität Hamburg, Martinistrasse 52, 20246 Hamburg, Germany
3. Experimental Neurophysiology, University Hospital Cologne, Joseph-Stelzmann-Str.
9, 50931 Köln, Germany; German Center for Neurodegenerative Diseases, LudwigErhard-Allee 2, 53175 Bonn, Germany (current address)
4. Institut für Physikalische Chemie, Universität Hamburg, Grindelallee 117, 20146
Hamburg, Germany
5. Institute of Histology and Embryology, School of Medicine, University of Belgrade,
Višegradska 26, 11000 Belgrade, Serbia
6. Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah,
Saudi Arabia
7. Center for Neuroscience, Shantou University Medical College, 22 Xin Ling Road,
Shantou,Guandong 515041, People’s Republic of China
1
#
These authors contributed equally to this work.
* Correspondence should be addressed to:
Rebecca Matsas, Laboratory of Cellular and Molecular Neurobiology, Hellenic Pasteur
Institute, 127 Vassilissis Sofias Avenue, 11521 Athens, Greece Tel: +30-210-64-78-843;
Fax: +30-210-64-78-833; e-mail: rmatsa@pasteur.gr
Melitta Schachner, Center for Neuroscience, Shantou University Medical College, 22 Xin
Ling Road, Shantou, Guandong 515041, People’s Republic of China. Tel: + 86-7548890-0276; Fax: + 86-754-8890-0276; e-mail: schachner@stu.edu.cn
2
Supplementary Information
Supplementary Methods
Neurite outgrowth and cell death assays in primary cultures of cerebellar neurons
Primary mouse cerebellar neurons were isolated from postnatal day 7 mouse cerebellum,
according to a previously described protocol.1 For estimation of neurite outgrowth, cells
were plated in poly-L-lysine (Sigma-Aldrich) coated 48-well plates at low density
(25,000 cells/cm2) in culture medium (Neurobasal supplemented with B27, N2, Glutamax
– all from Life Technologies – and KCl 25μM) containing PEG2000 (0.625% w/v) or
PEG-AuNP-40 (0.04 nM) or PBS vehicle (each treatment in quintuplicate). After 24 hrs
cells were fixed with 4% paraformaldehyde in PBS and stained with Toluidine
Blue/Methylene Blue1. Individual neurons that had extended neurites were captured
under a camera-equipped inverted light microscope and the length of their neurites was
measured using ImageJ. Only cells with neurites longer than twice their cell body
diameter and not in contact with other cells were taken into account. At least 100 cells
were measured for each condition.
To determine the neuroprotective effect of PEG-AuNPs, a cell death assay was
performed. Cerebellar neurons were seeded at 250,000 cells/cm2 in a 48-well-plate and
cultured for 24 hours. Cells were then treated with PEG2000 (1 µM) or PEG-AuNP-40
(0.04 nM) and after a 20 min incubation time at 37°C cell death was induced by addition
of 10 µM hydrogen peroxide. After a further 24-hour incubation at 37°C, live cells were
stained with Neutral Red (0.02%) for 30 minutes, washed with Hank’s Balanced Salt
3
Solution without Phenol red, lysed with 200 µl lysis buffer [1% (v/v) CH3CHOOH, 50%
(v/v) EtOH, 49% (v/v) distilled water] for 20 min at room temperature to release the
incorporated dye and absorption was measured at 540 nm.2 Signals obtained from
untreated cells were set to 100% survival and six wells were used for each condition.
Behavioral assessment of motor function recovery
Basso Mouse Scale (BMS) locomotor rating
Hind limb locomotor function was assessed using the Basso Mouse Scale (BMS)
rating.3,4 Locomotor performance of each animal was evaluated during free movement in
an open field. All lesioned animals were observed for 4 min, at 3 days and then every 2
weeks after the operation. Scoring was done blindly by two independent observers.
Single-frame motion analysis
Foot-stepping angle. Motor recovery was further analyzed quantitatively using a
numerical measure for plantar stepping evaluation, the foot-stepping angle.5 A left- and a
right-side view of each animal during two consecutive walking trials on a wooden beam
(1 m long, 4 cm wide) was captured before the operation and at various time points (3
days after the operation and every 2 weeks thereafter) with a digital video camera and the
acquired frames were analyzed using Virtual Dub software (version 1.6.19;
www.virtualdub.org) and ImageJ (version 1.48s; http://imagej.nih.gov/ij). The footstepping angle is defined by a line parallel to the dorsal surface of the hind paw and the
4
horizontal line. The angle is measured with respect to the posterior aspect at the
beginning of the stance phase. In uninjured mice, this phase is well defined and the angle
is around 20°. After SCI and severe loss of locomotor abilities, the mice drag behind their
hind limbs with dorsal paw surfaces facing the beam surface. The angle is increased to
>150°. Values for the left and right extremities were averaged.
Inclined ladder climbing test. Even without training, mice climb up rapidly an inclined
ladder with rare explorative stops and never turn back to descend the ladder. This
instinctive behavior is preserved even in severely disabled animals that climb, although
slowly, up to the top of the ladder using their forelimbs. The inclined position (55°) of the
ladder provides body weight support for disabled mice and thus aids climbing. The mice
were placed at the bottom rungs of the ladder and climbing was video recorded from a
position viewing the ventral aspect of the animals. The number of correct steps defined as
correct placing of the hind paw and sustained position until the next forward move, over
36 rungs were counted. Intact animals typically stepped on every second rung,
performing approximately 20 correct steps (averaged for the two extremities) to cross the
36-rung distance5.
Tissue processing
Tissue fixation and sectioning was performed as previously described.6-8 Mice were
anaesthetized with a 16% w/v solution of sodium pentobarbital (Narcoren, Merial,
Hallbergmoos, Germany, 5 μl g-1 body weight) and then transcardially perfused with
fixative (4% w/v paraformaldehyde and 0.1% w/v CaCl2 in 0.1 M cacodylate buffer, pH
5
7.4). Following perfusion, the spinal cords were left in situ for 2 hours at RT, after which
they were dissected out and post-fixed overnight at 4°C in the same fixative. Tissue was
then immersed in a 15% w/v sucrose solution in 0.1 M cacodylate buffer, pH 7.4, for 2
days at 4°C, embedded in Tissue Tek (Sakura Finetek, Zoeterwoude, NL), and frozen by
a 2 min immersion into 2-methyl-butane (isopentane) precooled to -80°C. Spinal cords of
each group were randomized into two groups, one for transverse and one for sagittal
sectioning. Serial 25 μm thick sections were cut in a cryostat and collected on SuperFrost
Plus glass slides. Sampling of sections was always done in a standard sequence so that
ten transverse or six parasagittal sections 250 μm apart were present on each slide. All
sections of one slide from every mouse were devoted to each histological parameter
checked.
Electron microscopy
Tissue was processed for electron microscopy as previously described9. Briefly, mice
were anaesthetized with sodium pentobarbital and perfused transcardially with a mixture
of 4% w/v paraformaldehyde and 2.5% v/v glutaraldehyde in 0.1 M cacodylate buffer
(pH 7.4). After overnight post-fixation in 4% w/v paraformaldehyde and 5% v/v
glutaraldehyde in the same buffer at 4oC, from each spinal cord, 1 cm long segments
were cut from the thoracic part at a distance of approximately 1 mm proximal and distal
to the center of the lesion. Tissue samples were post-fixed in 1% w/v osmium tetroxide in
0.1 M sodium cacodylate buffer, pH 7.4, for 1 h at room temperature, dehydrated and
embedded in epon resin according to standard protocols.10 Using a random sampling
6
protocol11, ultrathin sections were prepared on a Leica Ultracut UCT microtome and
stained with uranyl acetate-lead citrate. Electron micrographs were taken on a FEI
Morgagni 268D transmission electron microscope.
Immunohistochemistry
Indirect immunofluorescence was performed as previously described.6-8 Sections were
typically subjected to antigen retrieval in 10 mM sodium citrate solution, pH 9 (80°C, 30
min), followed by blocking of nonspecific sites and permeabilization with normal
serum/0.2% v/v Triton X-100 in PBS, for 1 h at RT, before the application of the primary
antibodies. For the detection of P0 myelin protein, sections were pre-treated for 5 min
with ice-cold methanol followed by incubation with goat polyclonal anti-P0 (1:100,
SantaCruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-CASPR/paranodin
(Contactin Growth Associated Protein, generous gift by Prof. D. Karagogeos). Rabbit
anti-glial fibrillary acidic protein (GFAP) (1:1,000; Dako Cytomation) or mouse antiGFAP (1:1,000; Sigma, St. Louis, MO) was used to detect astrocytes and visualize the
glial scar while rabbit anti-fibronectin (FN, 1:100; Sigma) was used to demarcate the
lesion epicenter. Goat anti-choline acetyltransferase antibody (ChAT) (1:100; Millipore,
Hofheim, Germany) was used to identify motor neurons and their cholinergic boutons in
the ventral horns of the lumbar spinal cord and mouse anti-vesicular GABA transporter
(VGAT) (1:1,000; Synaptic Systems, Goettingen, Germany) was used to detect the
perisomatic densities of GABAergic synapses on the cell bodies of the motor neurons, as
identified by their size and location. Rabbit anti-ionized binding calcium adapter
7
molecule 1 (Iba-1) (1:1,500; Wako Chemicals, Richmond, VA) stained microglial cells.
Rabbit polyclonal anti-neurofilament 200 (NF-200, 1:500, Sigma) was used to detect
neuronal axons while rat monoclonal anti-serotonin (5-HT, 1:50, Chemicon) was used to
identify serotonergic (5-hydroxytryptamine positive, 5-HT+) fibers and rabbit antityrosine hydroxylase (TH) (1:800; Chemicon) to identify catecholaminergic fibers. The
appropriate secondary antibodies conjugated to Alexa-Fluor photostable fluorescent dyes
(all from Molecular Probes) were used for visualization. Cell nuclei were labeled with
TO-PRO-3 and DAPI. Prolong Gold antifade curing mountant (Molecular Probes) was
used for mounting. Specimens were viewed under Leica TCS SP and Leica TCS-SP5II
confocal microscopes.
Motor neuron number, soma size and quantification of perisomatic terminals
Estimations of motor neuron numbers, soma areas and perisomatic terminals were
performed as described previously.5 Transverse sections of the spinal cord were serially
cut over a distance of 500 μm caudal to the caudal edge of the lesion site and were
stained for ChAT or VGAT. As ChAT immunoreactivity may decline in the cell soma
after injury, cell bodies of motor neurons we counted for large cellular profiles present in
the ventral horns of the spinal cord decorated with ChAT+ perisomatic synaptic puncta
and with varying degrees of, or no detectable, ChAT positivity in their cell somata (see
Fig. 5). In sections not labeled for ChAT, motor neurons were identified by their
clustered localization in the ventral horns of the spinal cord, the size of their somata in
combination with their large and hollow nuclei (unstained profiles) surrounded by
VGAT+ immunoreactive puncta. Longitudinal sections were also stained for ChAT to
8
measure motor neuron soma areas and perisomatic ChAT+ immunoreactive puncta.
Stacks of 1 μm-thick images were obtained on a Leica TCS SP confocal microscope
using a 40x objective and digital resolution of 1024 x 1024 pixels. Four adjacent stacks
(frame size, 250 x 250 μm) were obtained consecutively in a rostro-caudal direction so
that motor neurons located both close and remote (over a distance of 1,000 μm) to the
lesion site were sampled. One image per cell at the level of the largest cell body crosssectional area was used to measure soma area, perimeter, and number of perisomatic
terminals. Areas and perimeters were measured using ImageJ. Linear density was
calculated as number of perisomatic terminals per length unit of the cell perimeter.
Quantification of axonal sparing/regrowth
Parasagittal spinal cord sections immunostained for NF, TH, 5-HT were used to analyze
total fiber sparing/regrowth in the lesion site (NF), monaminergic (TH) and serotonergic
(5-HT) axons in the lumbar spinal cord. NF+ fibers crossing an arbitrarily selected border
perpendicularly spanning the lesion site and TH+ or 5-HT+ axons projecting beyond an
arbitrarily selected border 250 μm caudally to the lesion site were counted in every 10th
parasagittal serial section from the spinal cord and averaged per section per mouse.
Image processing and quantification of fluorescence intensity
For evaluation of expression levels of cell type-specific markers such as Iba-1, GFAP and
P0, fluorescence intensity was measured as pixel intensity on single channel stacks of
9
confocal images, acquired under the same settings. In particular, the laser power was set
to the lowest level that would be adequate for high signal/noise ratio acquisition and
would not bleach the specimen. Gain and offset were set to constant levels optimized so
as to avoid over- and under-exposure acquisition. Each series in the confocal stack was
averaged 3 times and the step size was 1 μm while image resolution was 1024 x1024
pixels. Optimization of acquisition settings was done using additional control sections not
included in the quantification to avoid endangering the experimental specimens with
fading. For quantification of P0 immunoreactivity, 6 sections were sampled per mouse
and in each section 4 fields were randomly selected within the area of interest and
scanned by a blinded experienced observer.
Image processing for quantification of fluorescence intensity was performed using
the ImageJ software, after selecting the region of interest on single channel stacks using
the free-hand selection tool and setting the threshold at a value that was kept constant
throughout, by a blind observer. Measurements were performed on each single image of
the confocal stack and then added up and normalized to the corresponding area. The
measurements from the 4 randomly selected fields (for Iba-1 and GFAP) were averaged
per section and the results from 6 sections were averaged per mouse. For P0 all fields
with internodes were processed and measurements were added up per section and
averaged per mouse. The results from all mice of a group were averaged per group.
Stereological analysis
10
Iba-1+ microglia were counted on an Axioscope microscope (Zeiss) equipped with a
motorized
stage
and
Neurolucida
software-controlled
computer
system
(MicroBrightField, Williston, Vermont, USA) using the optical dissector method as
described.9 Longitudinal spinal cord sections including the lesion site and approximately
2 mm-long segments rostral and caudal to the lesion center were used for counting. The
sections were observed under low-power magnification (10x objective) with a
365/420 nm excitation/emission filter set (01, Zeiss, blue fluorescence). Nuclear
counterstaining allowed for delineation of the spinal cord area. The numerical density of
Iba-1-immunoreactive cells was estimated by counting the nuclei of immunolabeled cells
within systematically randomly spaced optical dissectors. The parameters for this
analysis were guard space depth 2 μm, base and height of the dissector 3600 μm2 and
10 μm, respectively, distance between the optical dissectors 60 μm, using the objective
Plan-Neofluar 40x/0.75. Spinal cord areas 250 μm rostral and 250 μm caudal to the lesion
center were evaluated in 6 sections, each 250 μm apart. The counts were performed by
one observer in a blinded fashion.
Statistical analysis
Statistical analysis of motor behavior was performed using two-way ANOVA for
repeated measures with factors ‘treatment’ and ‘time’ followed by Holm-Sidak post hoc
test. Two-tailed Student’s t-test was used for comparison of the immunohistochemical
data at the end point of the experiment, where data from two groups were analyzed. Oneway ANOVA with Holm-Sidak post hoc was used for the in vitro experiments. P value
11
for significance was set to 0.05. All presented values are expressed as means ± standard
error of means (SEM).
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Supplementary Figure Legends
Supplementary Figure 1| Comparison between PEG on AuNPs and free PEG, in
vivo. a, b, Representative transmission electron microscopy images of PEG-AuNP-14
and PEG-AuNP-40 in vitro, respectively. Scale bar, 100 nm in a, 500 nm in b. c,
Representation of body weight reduction calculated for the four experimental groups of
mice. Mice of the PEG-AuNP-40 (n=6) and PEG (n=4) groups recover their weight more
efficiently than the PBS (n=5) and PEG-AuNP-14 (n=6) groups. Statistically significant
differences against the PBS group are indicated by asterisks of the corresponding color.
d, Locomotion rating in the open-field according to the Basso Mouse Scale, before
injury, early after injury (3 days) and then up to 6 weeks after injury with 2-week
intervals. A significant improvement of the PEG-AuNP-40 group is noted versus other
groups at 4 weeks. e, Foot-stepping angle results, as produced by single-frame motion
analysis on beam walking. No significant difference between groups was seen. f, Graph
representing the number of correct steps performed on the inclined ladder. The trend in
favor of PEG-AuNP-40 is significant at 6 weeks post injury as compared to the PBS
control and the PEG group. A lower level of significance was observed for the PEGAuNP-14 group versus the PEG and control groups. Values represent means ± SEM. *,
p≤0.05, **, p≤0.01 and ***, p≤0.001, by two-way ANOVA for repeated measures, with
Holm-Sidak post hoc test. For d, e, f the number of mice per group was the same as in c.
* is used for comparison of the PEG-AuNP-40 group (red) or the PEG-AuNP-14 group
(orange) with the PBS control group and # for comparison with the PEG group; ¤ shows
comparison of the PEG-AuNP-40 group with the PEG-AuNP-14 group.
13
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