nature methods | VOL.7 NO.11 | NOVEMBER 2010 | 905
which we appended a C-terminal (AU1) epitope (RevM10.AU1).
We injected intravenously 5 × 108 infectious units (IU) of
simian virus RevM10.AU1 (SV(RevM10.AU1)) or control simian
virus bilirubin-uridine 5′-diphosphate-glucuronosyl transferase
(SV(BUGT)) vectors, with or without prior intraperitoneal admin-
istration of mannitol in Balb/c mice. All injections were in 100 μl
of phosphate-buffered saline (pH 7.4) unless otherwise stated.
One month after injection of SV(RevM10.AU1), ~3% of all cells
expressed AU1 in mice that did not receive mannitol. Prior intra-
peritoneal inoculation of mannitol in SV(RevM10.AU1) recipients
resulted in an about tenfold increase in AU1-expressing cells
efficient cns gene delivery
by intravenous injection
Jean-Pierre Louboutin1, Alena A Chekmasova1,
Elena Marusich1, J Roy Chowdhury2 & David S Strayer1
We administered recombinant sV40-derived viral vectors
(rsV40s) intravenously to mice with or without prior
intraperitoneal injection of mannitol to deliver transgenes
to the central nervous system (cns). We detected transgene-
expressing cells (mainly neurons) most prominently in the
cortex and spinal cord; prior intraperitoneal mannitol injection
increased cns gene delivery tenfold. intravenous injection of
rsV40s, particularly with mannitol pretreatment, resulted in
extensive expression of multiple transgenes throughout the cns.
Viral gene transfer to the central nervous system (CNS) is of great
interest, but gene delivery to the CNS, especially diffuse delivery,
presents particular challenges. Peripheral vector administration
for transgene delivery to the CNS is potentially very useful for
many experiments in neurobiology. Although gene transfer to
the CNS of mammals is possible with several vectors1–3, in rare
cases via peripheral administration4, each system has limitations.
Expression of therapeutic proteins may also be useful in treating
several CNS disorders, but such approaches have so far yielded
very limited results5,6.
Recombinant large T antigen–deleted SV40-derived viral
vectors (rSV40s) deliver genes to nondividing cells efficiently
and achieve long-term transgene expression in vitro and in vivo7.
These vectors can transduce >95% of cultured human NT2 cells,
primary human neurons8 and microglia9 without detectable
toxicity. We have demonstrated previously that SV40-derived
vectors provide long-term gene expression in the brain, whether
administered locally by intraparenchymal administration or
diffusely by intraventricular administration, the latter with or
without intraperitoneal injection of mannitol10–13.
In some applications, generalized gene delivery throughout
the brain is desirable. Mannitol enhances transgene expression
by relaxing the blood-brain barrier. However, few studies report
the effects of prior injection of mannitol on CNS transgene
expression14,15. Our goal in this study was to test rSV40 gene trans-
fer to the CNS in vivo after intravenous vector administration,
with or without intraperitoneal mannitol to relax the blood-brain
barrier. We tested the transfer of several genes in these studies. We
initially used a rSV40 vector carrying RevM10, a dominant nega-
tive mutant of the HIV-1 transcript binding chaperone, Rev, to
1Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. 2Department of Medicine, Albert Einstein College of Medicine, Bronx,
New York, USA. Correspondence should be addressed to J.-P.L. (firstname.lastname@example.org).
Received 19 May; accepted 13 SepteMbeR; publiShed online 17 octobeR 2010; doi:10.1038/nMeth.1518
DAPI and AU1
Cells expressing AU1 (%)
Cells expressing AU1 (%)
Cells expressing AU1 (%)
Number of injections
1 × 108
5 × 108
1 × 109
figure 1 | AU1 expression from a transgene after intravenous
injection of recombinant SV40 virus. (a) AU1 immunostaining in
coronal cryostat sections of brains of mice injected intravenously
with SV(RevM10.AU1) or control SV(BUGT), with or without prior intra
peritoneal administration of mannitol. The motor cortex is shown. Scale
bar corresponds to 60 μm in the top three images and 30 μm in the
bottom image. (b–d) Percentages of AU1positive cells in the indicated
brain regions after indicated treatments (b), with different doses of
intravenously injected vector with prior mannitol treatment (c) and after
different numbers of injections of intravenous vector with prior mannitol
treatment of mice (d). Error bars, ± s.e.m. (n = 5 mice for each treatment
group). **P < 0.001. In c and d, unless indicated on the plots, P < 0.05
between the different doses (c) and between the numbers of injections (d).
© 2010 Nature America, Inc. All rights reserved.
906 | VOL.7 NO.11 | NOVEMBER 2010 | nature methods
(P < 0.001). We detected no expression of AU1 after intravenous
injection of SV(BUGT) (Fig. 1a,b). Most of the transgene express-
ing cells were in the motor and somatosensory cortex. We then
investigated the relationship between the dose of the vector and
the extent of transgene expression. We injected 1 × 108 IU, 5 × 108
IU and 1 × 109 IU of SV(RevM10.AU1) into mice with prior man-
nitol administration. The number of transgene-expressing cells
increased with higher doses of SV(RevM10.AU1) (Fig. 1c).
We determined whether multiple administrations of the
vector could increase the numbers of transgene-expressing
cells. We subjected mice to one, two or three injections of
1 × 109 IU of SV(RevM10.AU1) each one week apart after mannitol
administration. We collected brains four weeks after the first
injection (that is, 2 weeks after the third injection). Differences
in the numbers of transgene-expressing cells between mice given
one and several injections of SV(RevM10.AU1) were significant
in the different areas we examined (P < 0.05; Fig. 1d).
One month after injection of SV(RevM10.AU1), most of the
AU1-positive cells expressed the neuronal marker NeuN and
were stained by the fluorescent stain NeuroTrace (Fig. 2a–c). Few
NeuroTrace-stained cells expressed AU1 in the mice that did not
receive mannitol, but we observed many AU1-positive cells in the
group with prior injection of mannitol (Fig. 2a). Occasionally we
observed transgene-expressing cells immunopositive for Iba-1,
a marker of microglial cells (Fig. 2d). We observed no immuno-
positivity for AU1 in astrocytes (Fig. 2e).
Transgene expression was widely distributed in the cortex
in the mice that received mannitol (Fig. 3a). We observed the
majority of AU1-positive cells in different cortex areas (cingulate,
motor, somatosensory, piriform and visual). We observed fewer
transgene-expressing cells in ventral hippocampus, caudate
putamen and lateral septum. We observed rare transgene-
expressing cells in the molecular layer of the dorsal hippocampus
and in the molecular and granular cells layers of the cerebellum
(Fig. 3b,c), and comparatively few transgene-expressing cells in
the basal ganglia or in the cerebellum after injection of 1 × 108 IU
or 5 × 108 IU of SV(RevM10.AU1) (data not shown).
We also observed transgene-expressing cells in the cervical,
thoracic and lumbar spinal cord, and expression was widespread
in the posterior and anterior horns (Fig. 4a). In the group given
a prior injection of mannitol, we observed more transgene-
expressing cells (Fig. 4b). AU1-positive cells were mostly neurons,
some with the morphology and size of sensory or motor neurons,
others of interneurons (Fig. 4c).
We then used this approach to investigate the delivery of
additional transgenes to different areas of the brain. We tested
the delivery of potential therapeutic transgenes, Cu/Zn super-
oxide dismutase (Sod1) and glutathione peroxidase (Gpx1) with
figure 2 | Transgeneexpressing cells were mostly neurons. (a) Cryostat
sections of mice brains (motor cortex is shown) immunostained for AU1
and stained with NeuroTrace one month after intravenous injection with
the indicated vectors with or without prior intraperitoneal mannitol
injection. Arrows point to neurons expressing AU1. Insets show higher
magnification images. Scale bar corresponds to 60 μm in large images and
15 μm in insets. (b) Higher magnification of another field of the cryostat
section shown in the bottom image in a. (c–e) Representative images of
sections from mice injected with SV(RevM10.AU1) and prior intraperitoneal
mannitol administration, immunostained for NeuN and AU1 (c), Iba1 and
AU1 (d), and GFAP and AU1 (e). Five mice were examined in each treatment
group. Data are representative of three experiments. Scale bars, 10 μm (b),
20 μm (c), 25 μm (d) and 40 μm (e).
Cingulate cortexMotor cortex Somatosensory cortexVisual cortex Dorsal hippocampus
Ventral hippocampusCaudate putamen Lateral septum Cerebellum Medial vestibular nucleus
Cells expressing AU1 (%)
figure 3 | Distribution of AU1 expression
in the brain. (a) Image of a cryostat
section through the cingulate cortex of
mice one month after injection of viral
vectors with prior mannitol treatment.
Sections were labeled with DAPI (left)
and immunostained for AU1 (right).
M1, primary motor cortex; M2, secondary
motor cortex; CG, cingulate cortex; PrL,
prelimbic cortex; IL, infralimbic cortex;
Pir, piriform cortex; LO, lateral orbital cortex;
and AI, agranular insular cortex. (b) Images
showing transgene expression (AU1, green)
in the indicated brain areas overlaid with
NeuroTrace staining (red). DG, dentate gyrus;
Mol, molecular layer of the dentate gyrus;
ML, molecular layer of the cerebellum;
PCL, Purkinje cell layer; and GCL, granule
cell layer. (c) Percentages of total cells
(DAPIstained) and of neurons (NeuroTrace
stained) expressing AU1 in the indicated
areas of the brain. Error bars, s.e.m. (n = 5
mice in each treatment group). Scale bars,
240 μm (a) and 30 μm (b).
Neurotrace and AU1
GFAP and AU1
© 2010 Nature America, Inc. All rights reserved.
nature methods | VOL.7 NO.11 | NOVEMBER 2010 | 907
prior mannitol administration. Again, we found the major-
ity of SOD1- and GPx1-expressing cells in neurons in different
areas of the cortex (Fig. 4d,e and Supplementary Figs. 1 and 2).
We also observed SOD1 expression in the substantia nigra
(Supplementary Fig. 1). To expand the scope of our studies, we
tested delivery of SV(Nef-Flag) using this approach. We observed
the majority of transgene-expressing cells in neurons in the motor
and somatosensory cortex, with fewer transgene-expressing cells
in other regions of the brain (Fig. 4f and Supplementary Fig. 3).
The transduction efficiencies were similar in the same areas of the
brain for all four vectors we used. All vectors were comparable in
the regions and cell types in the brain that expressed transgenes
upon intravenous administration after intraperitoneal injection
We observed no inflammation or increase in the number of
microglial cells in the different sections of the brains of mice injected
with rSV40 vectors, with or without prior mannitol administration
(Supplementary Fig. 4a,b). There was no detectable AU1 expres-
sion in liver, kidney, heart and skeletal muscle (Supplementary
Fig. 4c). This suggests that the prior inoculation of mannitol
focused gene delivery to favor the CNS because routine intravenous
inoculation of rSV40s will transduce the liver and kidney7.
Notably, rSV40 vectors transduced both neurons and micro-
glia, the two main CNS populations. AAV9 injected intra-
venously can bypass the blood-brain barrier4 but preferentially
targets neonatal neurons and astrocytes4, whereas in our
current study rSV40s injected intravenously targeted mainly
mature neurons. Some marker genes, such as the fluorescent
proteins or β-galactosidase, are not expressed well when deli-
vered by rSV40 vectors packaged by COS-7 cells. The reasons for
this are not yet known but appear to reflect a process that occurs
during packaging in a transgene-specific fashion. Sometimes,
this limitation has been circumvented by using a different pro-
moter7. Other mammalian DNAs are largely expressed effec-
tively by these vectors. Our results nonetheless suggest that, for
most genes, delivery to substantial percentages of neurons in the
brain and spinal cord is possible using one or more intravenous
inoculations of rSV40s after mannitol administration.
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/naturemethods/.
Note: Supplementary information is available on the Nature Methods website.
We thank J. Altemus for technical help. Funding was provided by the US National
Institutes of Health (MH70287, MH69122 and AI48244 to D.S.S., DK68216 to J.R.C.).
J.P.L. designed and performed experiments, processed and analyzed data,
and wrote the paper. A.A.C. performed experiments and designed vectors. E.M.
designed vectors. J.R.C. analyzed data and provided collaboration and grant
support. D.S.S. coordinated the project and helped to write the paper.
comPeting financial interests
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturemethods/.
reprints and permissions information is available online at http://npg.nature.
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DAPl and AU1NeuroTrace and AU1
figure 4 | Expression of transgeneencoded AU1 in the spinal cord.
(a) Cryostat section through upper thoracic spinal cord of mice one month
after injection of viral vectors with prior mannitol treatment. Section was
labeled with DAPI (left) and immunostained for AU1 (right). DH, dorsal
horn; VH, ventral horn; and CC, central canal. Scale bar corresponds to
240 μm in large images and 120 μm in insets. (b) Images of AU1expressing
cells in the upper thoracic cord under the indicated conditions. Scale bar,
60 μm. (c) AU1immunolabeled and NeuroTracestained sections through
the thoracic and lumbar spinal cord. The shown scale bar corresponds to
60 μm for the top images and 45 μm for the middle and bottom images.
Some AU1expressing neurons had the morphology and size of sensory or
motor neurons (arrowheads), others of interneurons (arrows). Data are
representative of three experiments with five mice in each group.
(d–f) Percentages of total cells (DAPIstained) and of neurons (NeuroTrace
(NT)stained) expressing the transgenes SOD1 (d), GPx1 (e) and Flag (f) in
different areas of the brain. Error bars, s.e.m. (n = 5 mice in each group).
© 2010 Nature America, Inc. All rights reserved.
Animals. Adult male Balb/c mice (3 months old) were purchased
from Charles River Laboratories. Protocols for injecting and
killing mice were approved by the Thomas Jefferson University
Institutional Animal Care and Use Committee (IACUC), and are
consistent with Association for Assessment and Accreditation of
Laboratory Animal Care (AAALAC) standards. The diet that the
mice received was a regular powdered rodent diet without any
component that might cause oxidative stress (that is, high-fat diet
or high-manganese diet) and was not folate/methyl or iron defi-
cient. The mice had free access to water and food.
Antibodies. The following primary antibodies were used: flu-
orescein isothiocyanate (FITC)-conjugated mouse anti-AU1
(IgG2a; 1:100 dilution), mouse anti-AU1 (IgG3; 1:100) (Covance),
mouse anti-NeuN (IgG1; 1:100), a neuronal marker (Chemicon
International), rabbit anti-ionized calcium binding adaptor
molecule 1 (Iba1) (IgG; 1:100), a marker of quiescent and active
microglia (Waco Chemicals), mouse anti-glial fibrillary acidic
protein (GFAP) (IgG2b; 1:100) (Becton Dickinson), goat anti-
GFAP (1:100) (Santa-Cruz), rabbit anti-human Cu/Zn superoxide
dismutase (SOD1) (1:100) (Assay Designs), mouse anti-tyrosine
hydroxylase (TH) (IgG1; 1:100) (Immunostar), FITC-conjugated
mouse anti-Flag (IgG1; 1:100), mouse anti-Flag (IgG1; 1:100), rab-
bit anti-Flag (1: 100) (Sigma) and mouse anti-GPx1 (IgG1 kappa;
1:100; Abnova). The following secondary antibodies were used at
1:100 dilution: FITC and tetramethyl rhodamine isothiocyanate
(TRITC)-conjugated goat anti-mouse, TRITC-conjugated goat
anti-rabbit, FITC-conjugated sheep anti-rabbit (Sigma), FITC
and TRITC-conjugated donkey anti-mouse and anti-rabbit, and
FITC- and Cy3-conjugated donkey anti-goat antibodies (Jackson
Vector production. The general principles for making recom-
binant, large T antigen–deleted, replication-defective SV40 viral
vectors have been previously reported16–19. Uniquely among
gene transfer vectors, SV40 genome is circular and therefore
lacks terminal repeat regions that characterize many other
viral vectors with linear genomes. SV40 early promoter (EP)
and late promoters (LPs) are on opposite strands. The SV40-EP
drives expression of large T antigen (Tag) (responsible for
virus genome replication of wild-type SV40) and small t anti-
gen (tag). Beginning with the wild-type SV40 genome, the Tag
gene was replaced with a polylinker gene, downstream from the
SV40 EP. Occasionally, the late genes were also deleted. These
operations created space in the genome and as well made the
virus replication-crippled. A transgene with additional pro-
moter, enhancer, poly(A) signal and other sequences may be
cloned into the polylinker. Most of the constructs used carry
a polylinker downstream from the EP and retain the viral late
genes intact. The recombinant SV40 vectors are produced from
cloned vector genomes by removing the carrier plasmid (usually
pT7Blue or pGEM), gel-isolating and recircularizing the rSV40
genome, then transfecting into COS-7 cells. These cells carry an
integrated copy of the wild-type SV40 genome that is defective
at the origin of replication (and so cannot produce virus on their
own). They supply Tag in trans and supply all the viral proteins
needed to package the virus. The rSV40 that is produced in this
fashion is prepared from lysates of these transfected COS-7 cells.
rSV40 is then amplified by infecting COS-7 cells with rSV40
virus. No helper virus or additional transfection is used.
The purification process involves treatment of lysates of COS-
7 cells with deoxycholate, disaggregation of virus particles and
increases rSV40 yield compared to unpurified preparations.
It is routinely possible to attain purified virus yields greater than
1011 IU ml−1. Briefly, 3.5 × 106 infected cells were collected in
10 ml of cell debris and media, then both cells and media were
subjected to three cycles of freezing and thawing to break cell
membranes and free the viruses. One milliliter of 10% Triton
X-100 and 5% sodium deoxycholate was added to dissociate the
virus from cell membranes, and a Dounce homogenizer was
used to disaggregate cell membranes. Cell debris was removed
by centrifugation at 16,000g for 20 min. The virus present in
the supernatant was concentrated in a discontinuous sucrose
gradient (1.5 ml of 75% sucrose and 2.5 ml of 20% sucrose) by
centrifugation for 3.5 h at 74,000g. Then 0.5-ml fractions were
collected, pooled and dialyzed against PBS overnight at 4 °C
using sterile productions.
Replication-defective SV40 viruses can be titered by quantitative
PCR, which measures the number of rSV40 genomes in purified
viral stocks using primers specific for the rSV40 genome, coupled
with SYBR Green detection of PCR products. For SV(RevM10.
AU1), expression of the RevM10.AU1 transgene is driven by the
cytomegalovirus immediate early promoter (CMV-IEP). Sod1 and
Gpx1 transgenes were subcloned into pT7(RSVLTR), in which
transgene expression is controlled by the Rous sarcoma virus long
terminal repeat (RSVLTR) as a promoter. rSV40 viral vectors, car-
rying cDNA encoding HIV Nef protein with a C-terminal Flag
epitope tag with a cytomegalovirus immediate early promoter
(CMV-IEP) were also used. Briefly, to make vectors, the cloned
rSV40 genome was excised from its carrier plasmid, gel-purified
and recircularized, then transfected into COS-7 cells. These cells
supply in trans Tag and SV40 capsid proteins, which are needed to
produce recombinant replication-defective SV40 viral vectors20.
Crude virus stocks were prepared as cell lysates, then band-
purified by discontinuous sucrose density gradient ultracentri-
fugation and titered by quantitative PCR (Q-PCR; Stratagene)21.
As a control vector, we used SV(BUGT), which carries the cDNA
for human bilirubin-uridine 5′-diphosphate-glucuronosyl-
Intravenous injection of SV(RevM10).AU1. SV(RevM10.AU1),
or a control SV40 vector, SV(BUGT), were injected intravenously
(i.v.) by tail vein injection in mice (n = 5 in each group). For the
mannitol study, 3 ml of sterile 25% mannitol in 0.9% saline per
100 g body weight was injected intraperitoneally (i.p.) 20 min
before i.v. injection of SV(RevM10.AU1) (n = 5). Control mice
received saline i.p. instead of mannitol (n = 5). Then 5 × 108 IU
of SV(RevM10.AU1) or control viral vector, SV(BUGT), in 100 μl
phosphate buffer saline (PBS) were injected i.v. The brains and
the spinal cords of mice were examined 4 weeks after the injection
of the vector. Livers, kidneys, hearts and skeletal muscles (quadri-
ceps) were also examined for AU1 expression four weeks after
vector injection to study whether gene expression was observed
in different organs at this time point.
Injection of different doses of SV(RevM10.AU1). To investigate
the relationship between the dose of the vector and the transgene
© 2010 Nature America, Inc. All rights reserved.
nature methods Download full-text
expression, 1 × 108 IU, 5 × 108 IU and 1 × 109 IU of SV(RevM10.
AU1) in 100 μl PBS were injected i.v. with prior i.p. mannitol
administration (n = 5 in each group, total = 15). Brains were col-
lected 4 weeks later. Livers, kidneys, hearts and skeletal muscles
(quadriceps) were also examined for AU1 expression 4 weeks
after vector injection.
Multiple administrations of the vector. To study if multiple
administrations of the vector could increase transgene expression,
one, two or three i.v. injected doses of 1 × 109 IU of SV(RevM10.AU1)
in 100 μl PBS each one week apart were administered with
prior i.p. injected mannitol administration (n = 5 in each group,
total = 15). Brains were collected 4 weeks after the first injection.
Injection of SV(SOD1), SV(Nef-Flag) and SV(GPx1). Distri-
bution of Sod1, Flag and Gpx1 transgenes were assessed by inject-
ing intravenously either 1 × 109 IU of SV(SOD1), 5 × 108 IU of
SV(Nef-Flag) or 5 × 108 IU of SV(GPx1), in 100 μl PBS with
prior i.p. injected mannitol administration. Brains were collected
4 weeks later (n = 5 in each group, total = 15).
Immunocytochemistry. For immunofluorescence, coronal cryo-
stat sections (10 μm thick) were processed for indirect immuno-
fluorescence. Blocking was performed by incubating 60 min with
10% goat or 10% donkey serum in PBS (pH 7.4). Then, sections
were incubated with antibodies diluted according to manufac-
turer’s recommendations: 1 h with primary antibody, then 1 h
with secondary antibody diluted 1:100, all at room temperature
(20–22 °C). Double immunofluorescence analysis was per-
formed as previously described23. All incubations were followed
by extensive washing with PBS. Mounting medium contained
4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories)
to stain nuclei. Specimens were finally examined under a Leica
DMRBE microscope. Negative controls consisted of preincuba-
tion with PBS and 0.1% BSA, substitution of nonimmune isotype-
matched control antibody for primary antibody and/or omission
of the primary antibody.
Staining of neurons using NeuroTrace. To stain neurons, we
used NeuroTrace (NT; Invitrogen), a fluorescent stain that has
been used as a neuronal marker in many studies focusing on the
characterization of neurons24,25. After rehydration in 0.1 M PBS
(pH 7.2), sections were treated with PBS plus 0.1% Triton X-100
10 min, washed twice for 5 min in PBS then stained by NT (1: 100)
for 20 min at room temperature. Sections were washed in PBS
plus 0.1% Triton X-100 then × 2 with PBS, then let stand for 2 h at
room temperature in PBS before being counterstained with DAPI.
Combination NT and antibody staining was performed using pri-
mary and secondary antibodies staining first (see above), followed
by staining with NT. Experiments were repeated 3 times and were
done the same day for the different sections considered.
General morphology. Microscopic morphology of the brain
was assessed by hematoxylin-eosin staining of cryostat sections.
Hematoxylin-eosin staining was used to characterize a putative
inflammation in the brains of the four groups of mice injected
with the different vectors.
Morphometry. A computerized imaging system (Image-Pro Plus,
MediaCybernetics) was used to quantitate transgene-positive cells
as well as microglial cells immunopositive for Iba1. Transduction
was assessed for each brain 4 weeks after i.v. injection by serial
cryo-sectioning of the whole brain (10 μm thick coronal sections),
each slide being numbered, then by immunostaining of every
tenth section for AU1, SOD1, Flag and GPx1 (that is, at 100-μm
intervals). Between 100 and 120 sections were immunostained for
each mouse. AU1-positive cells were enumerated in the caudate
putamen, somatosensory and motor cortex in the experiments
concerning the titers and multiplication of injections. AU1-,
SOD1-, Flag- and GPx1-positive cells were enumerated in the
areas of interest in the study focused on the distribution of the
transgene. The total number (and not the number in random
areas) of AU1 positive cells in the whole area of interest for every
tenth section was counted and summed. The final number pre-
sented was an average of the results measured in the different
sections examined. The total number of positive cells in a brain
could be estimated by ‘multiplying’ the cell counts by the length
of the transduced area, assuming that the number of positive cells
in the sections spanned between the first and tenth section, for
example, will be close to the one measured on these first and
tenth sections. This procedure already described for assessment of
numbers of transgene-expressing cells in the brain26 allows quan-
titative and relative comparisons among different time points,
although it does not reflect the total number of transduced cells
in vivo. However, if these calculations of cell counts did not con-
form to unbiased stereological method, they were realized on
10-μm-thick sections and the total number of positive cells was
counted in each section, whereas stereological methods usually
analyze random fields of the sections. Moreover, we used our
current method to evaluate total numbers of neurons in the rat
normal striatum using immunocytochemistry (using anti-NeuN)
and to stain neurons by NT. Preliminary results showed a total
number of neurons close to the one measured using the optical
fractionator method (data not shown).
Statistical analysis. Results were expressed as average ± s.e.m.
Comparison of medians between two groups was achieved by
using the two-tail Mann-Whitney test. Comparison of medians
between more than two groups was done by using the Kruskall-
Wallis test. We used the Fisher’s exact test (with a two-sided
P value) for comparison of two percentages. Differences between
groups were considered significant for P < 0.05.
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