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The Journal of Neuroscience, February 15, 1999, 19(4):1335-1344
Suppression of Postischemic Hippocampal Nerve Growth Factor
Expression by a c-fos Antisense Oligodeoxynucleotide
Jian-Kun
Cui1,
Chung Y.
Hsu2, and
Philip K.
Liu1
1 Department of Neurosurgery, Baylor College of
Medicine, Houston, Texas 77030, and 2 Department of
Neurology, Washington University, St. Louis, Missouri 63110-1093
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ABSTRACT |
We examined the uptake and distribution of an antisense
phosphorothioated oligodeoxynucleotide (s-ODN) to
c-fos, rncfosr115, infused into the
left cerebral ventricle of male Long-Evans rats and the effect of this
s-ODN on subsequent Fos, NGF, neurotrophin-3 (NT-3), and actin
expression. To establish the uptake and turnover of s-ODN in the brain,
we studied the copurification of the immunoreactivity of biotin with
biotinylated s-ODN that was recovered from different regions of
the brain. A time-dependent diffusion and the localization of s-ODN
were further demonstrated by labeling the 3'-OH terminus of
s-ODN in situ with digoxigenin-dUTP using terminal
transferase and detection using anti-digoxigenin IgG-FITC. Cellular
uptake of the s-ODN was evident in both the hippocampal and cortical regions, consistent with a gradient originating at the ventricular surface. Degradation of the s-ODN was observed beginning 48 hr after
delivery. The effectiveness of c-fos antisense s-ODN was demonstrated by its suppression of postischemic Fos expression, which
was accompanied by an inhibition of ischemia-induced NGF mRNA
expression in the dentate gyrus. Infusion of saline, the sense s-ODN,
or a mismatch antisense s-ODN did not suppress Fos expression. That
this effect of c-fos antisense s-ODN was specific to NGF
was demonstrated by its lack of effect on the postischemic expression
of the NT-3 and  -actin genes. Our results demonstrate that
c-fos antisense s-ODN blocks selected downstream events
and support the contention that postischemic Fos regulates the
subsequent expression of the NGF gene and that Fos expression may have
a functional component in neuroregeneration after focal cerebral ischemia-reperfusion.
Key words:
antisense DNA; experimental cerebral ischemia; c-fos; drug target validation; gene regulation; gene
function analysis; immediate early genes; intracerebroventricular
delivery; neurotrophin; NGF; oligodeoxynucleotide; transfection; stroke
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INTRODUCTION |
The expression of the immediate
early genes (IEGs) (such as c-fos and c-jun),
followed by the expression of the late effector genes, such as the
neurotrophin genes (NTGs), in the ischemic brain raises the possibility
that IEG expression may play a role in the subsequent expression of
NTGs (Sharp, 1994 ). The expression of NTGs [e.g., nerve growth factor
(NGF) and brain-derived neurotrophic factor] may be important in
regenerative processes and functional recovery after stroke (Lindvall
et al., 1992; Ghosh et al., 1994). However, the pathophysiological
significance of the postischemic expression of the IEGs remains to be
fully elucidated (Sagar, 1993; Akins et al., 1996). In eukaryocytes,
gene expression is regulated primarily through sequence-specific
transcription factors. IEG products, such as Fos and Jun, form
heterodimers that bind specifically to the AP-1 site and are nuclear
regulatory proteins. The AP-1 binding sequence has been identified in
the promoter region of a number of genes, including NGF (Zheng and
Heinrich, 1988). A causal link between the activation of
c-fos and the subsequent expression of NGF has been found
in vitro (Hengerer et al., 1990), although the reverse
sequence of events had been reported earlier (Kruijer et al., 1985).
Further clarification of the relationship between c-fos and
NGF expression in the brain is needed.
We have shown that in vivo Fos-AP-1 knockdown is feasible
using a c-fos antisense phosphorothioated
oligodeoxynucleotide (s-ODN), which selectively blocks the translation
of the target c-fos mRNA (Liu et al., 1994). The commonly
used antisense s-ODNs for manipulation of gene expression are
single-stranded s-ODN, limited to 15 and 30 nucleotides in length for
an effective uptake by endocytosis (Yakubov et al., 1989). Although the
stability, disposition, and clearance of ODNs within the CSF of
the rat have been described (Whitesell et al., 1993), the parenchymal
uptake and distribution of ODNs within the brain are primarily
unexplored. Our decision to use an s-ODN to c-fos to explore
the regulation of gene expression in the brain was based on several
factors: (1) the possible involvement of AP-1 in apoptosis initiated by
tumor necrosis factor- (Roulston et al., 1998); (2) the robust
expression of c-fos after ischemic or traumatic brain injury
(An et al., 1993); (3) the well established methodology for a detection
of regional expression of Fos in the brain (Sagar et al., 1988; Sharp
et al., 1991); (4) the effective selection of ODN sequences using
in vitro translation and immunoprecipitation (Liu et al.,
1994); and (5) the ability to assess in vivo Fos function
based on its AP-1 binding activity in the brain extract. Moreover, many
laboratories have successfully applied c-fos antisense ODNs
in animal models (Funato et al., 1992; Chiasson et al., 1992, 1994;
Heilig et al., 1993; Dragunow et al., 1994; Hooper et al., 1994;
Schlingensiepen et al., 1994; Simonson, 1994; Cirelli et al.,
1995a; Quercia and Chang, 1996; Yu et al., 1996), which will allow us
to compare our results with those of these other laboratories. In this
study, we demonstrate the location of s-ODN and study the effects of
s-ODNs.
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MATERIALS AND METHODS |
s-ODN and preparation of biotinylated s-ODN
The s-ODNs to c-fos, including the following, were
custom made by National Biosciences, Inc. (Plymouth, MN): sense
orientation (sense-rncfosr115)
5'-ggtttgcccaaaccacgaccatgatg-3'-OH; the antisense orientation
(antisense-rncfosr115)
5'-catcatggtcgtggtttgggcaaacc-3'-OH; the same
antisense-rncfosr115 with the 3'-OH terminus blocked with
one dideoxynucleotide (s-ODN-ddC); and the mismatch MA17 s-ODN
5'-gaacatcatCgtGgCgg-3'-OH. The mismatch MA17 s-ODN had three
mismatching nucleotides and contained only 14 matching nucleotides to
the c-fos gene. Unless otherwise specified, the s-ODNs and biotinylated s-ODN (bio-s-ODNs) we used in this study were in the antisense orientation. The sense s-ODNs and the mismatch s-ODN to
c-fos were used only in selected control experiments to
confirm the specific effects of the antisense s-ODN. The s-ODN stock
was diluted to a final concentration of 1 nmol/µl saline. The
antisense s-ODN to c-fos was labeled using photobiotin
acetate according to the protocol described by Life Technologies
(Gaithersburg, MD). The resultant bio-s-ODN was purified using a
Sephadex G-25 column (Boehringer Mannheim, Indianapolis, IN) to remove
the unconjugated photobiotin acetate and stored at  20°C.
To verify that the length of the purified bio-s-ODN was the same as the
original c-fos antisense s-ODN (26 nucleotides), the bio-s-ODN was labeled with [ 32P]ATP (New England
Nuclear, Boston, MA) using T4 polynucleotide kinase
(Promega, Madison, WI) and then resolved using PAGE (16%; Bio-Rad, Richmond, CA). The concentration of the bio-s-ODN was measured
using a colormetric detection kit and the protocol specified by the
manufacturer (Life Technologies). The pixel values of the biotin spots
on the blot were quantified using an image analyzer (IS2000; Alpha
Innotech Corp., San Leandro, CA).
Delivery of s-ODNs
One hundred ninety-one Long-Evans hooded male rats (body weight
300 ± 30 gm) from Harlan (Indianapolis, IN) were used in the entire study. Housing and anesthesia concurred with guidelines established by the Institutional Animal Welfare Committee in accordance with the Public Health Service Guide for the Care and Use of
Laboratory Animals, United States Department of Agriculture
regulations, and the American Veterinary Medical Association Panel on
Euthanasia guidelines. Anesthesia was induced with ketamine (100 mg/kg, i.p.) plus xylazine (10 mg/kg, i.p.). A total of 40 µl of
artificial CSF (aCSF) containing lipofectin (10 µg, unless otherwise
indicated) and one of various s-ODNs (as specified in the text) was
infused over 30 sec into the left lateral ventricle guided by a
stereotaxic device (Liu et al., 1994). In lipofectin controls,
lipofectin without s-ODNs (n = 3) in 40 µl of aCSF
was infused in the same manner. In other controls, saline
(n = 16), s-ODN alone in aCSF (n = 9),
or animals without surgery (n = 5) (saline, S-ODN minus lipofectin, or normal controls; respectively) was delivered. At specified times after delivery, the animals were anesthetized and tissue fixation (s-ODN uptake and recovery studies) or for the surgery to produce experimental stroke (Fos and neurotrophin gene
expression studies). All experiments were performed in a blinded
manner, with the rats and s-ODN or control treatments randomly
administered to minimize any selection, treatment, or observation bias
on the part of investigators. Unless specified, at least three animals
were used for each treatment in the rat brain.
Tissue preparations
The animals were anesthetized if needed at the time of death
using transcardial perfusion with 150 ml of saline at a rate of 40 ml/min, followed by 300 ml of 4% paraformaldehyde in 0.1 M
phosphate buffer (PB), pH 7.4, at a rate of 20 ml/min. The brain was removed, and 3 mm coronal sections were prepared. The brain slices
were incubated twice in 0.01 M PB for 1 hr and then in a
series of increasing concentrations of ethanol (70-100%) in 0.01 M PB before being embedded in paraffin. For in
situ 3' end labeling of s-ODN, coronal brain sections (5 µm) from paraffin-embedded brains were treated with xylene and
then with chloroform plus xylene. For immunohistochemistry and in
situ hybridization (ISH), the brain tissue was not embedded in
paraffin but was stored as frozen sections (20-µm-thick on
poly-L-lysine-coated slides). In all examinations, two to
four brain slices from each animal were mounted on a
poly-L-lysine-coated slide, and each slice was obtained
~0.2 mm apart, located between 3.6-4.8 mm from bregma. For dot blot
analysis of s-ODN recovery or for gene expression using reverse
transcription-PCR (RT-PCR), the brains without perfusion were removed
at the designated times. The cortex, hippocampus, and cerebellum were
separated, flash-frozen in liquid nitrogen, and stored at 75°C (Liu
et al., 1994).
In situ uptake and distribution of s-ODNs
In our studies described here, three complementary assays were
used for the pharmacokinetic study of s-ODN uptake in the right hemisphere. These include a direct immunohistochemical staining of the
bio-s-ODNs and an in situ 3'-OH-terminus labeling of s-ODNs. Moreover, the length of the recovered bio-s-ODN was purified using Sephadex G-25 column-recovered bio-s-ODNs and Centricon-10 (Amicon, Beverly, MA).
Direct detection using the immunohistochemical method. The
objective of this study was to determine the optimal time during which
the brain took up bio-s-ODNs in 14 animals that received the antisense
bio-s-ODN. The presence of bio-s-ODN in the brain tissue was detected
using the primary antibody (goat anti-biotin IgG; Sigma, St. Louis,
MO), which was recognized using a secondary antibody (rabbit anti-goat
IgG-FITC; Sigma). Briefly, the brain slices were incubated with the
primary antibody (1:800) for 16 hr at 4°C, washed with PBS
three times, and then incubated with the secondary antibody (1:1600)
for 2 hr at room temperature. After washing in PBS, the DNA in the
nuclei was stained using propidium iodide (PI) at a concentration of
0.5 µg/ml in the presence of heat-inactivated RNase A (0.5 µg/ml;
Stratagene, La Jolla, CA) for 5 min. The orange-red coloration of the
DNA caused by PI staining serves as an indicator for the
location of the nuclei. Each brain slice was examined for the antibody
FITC under a microscope using a Leica (Nussloch, Germany) I3
filter (450-510 nm) and a mercury light source. Photographs of the
right cerebral hemisphere were acquired using either a regular camera
with Kodak type film (Eastman Kodak, Rochester, NY) or a cooled color
digital camera (the SPOT camera; Diagnostic Instruments, Sterling
Heights, MI), with the digitized image stored on a computer diskette.
The stability of biotin signals on bio-s-ODNs was determined by
detecting biotin label in the bio-s-ODN from the brains of eight
animals at 29, 48, and 72 hr after delivery. The assay was designed to
measure biotin on the recovered s-ODNs. Nuclear DNA from cerebral
cortex, hippocampus, and cerebellum was extracted (Liu et al., 1994).
One-fifth of each DNA sample (or 100 µl) was processed through the
Sephadex G-25 Quick-Spin column to recover the bio-s-ODN of greater
than 20 bases. Further dialysis of the column-recovered DNA in a
Centricon-10 membrane (Amicon) detected no residual bio-s-ODN in the
flow-through, indicating that the bio-s-ODN was 26 bases. The Sephadex
G-25 column-purified bio-s-ODN was then vacuum-dried and resuspended in
10 µl of STE buffer [(in mM) 50 NaCl, 10 Tris-HCl, and 1 EDTA], and one-fifth (2 of 10 µl) of the
column-purified fraction was applied onto blotting paper for the
measurement of biotin signal. Of the recovered bio-s-ODN, a set
of 2-10 pg bio-s-ODN standards (before column purification) and a set
of recovered bio-s-ODN after column purification were also applied onto
the blotting paper in the second row. The amount of bio-s-ODN was
measured using the colormetric detection kit stated above. The
detection limit using this method was 250 pg of bio-s-ODN from the
brain sample.
In situ labeling of 3'-OH terminus of the s-ODN. The
objective of this study was to determine the distribution of s-ODN in the brain tissue. Seventy-nine animals were studied (n = 3 each that received 1 and 50 nmol, and n = 2 in 100 nmol antisense c-fos s-ODN; n = 19 in
the group that received 10 nmol of antisense c-fos s-ODN;
n = 20 for s-ODN-ddC; and n = 15 for
those that received sense c-fos s-ODN). The 3'-OH terminus
on s-ODN in the brain can be labeled using terminal transferase (TdT)
in the presence of digoxigenin (dig)-dUTP, followed by the
antibody against digoxigenin with FITC conjugates. TdT synthesizes a
DNA chain by 5' to 3' polymerization using 5'-dNTP (or dig-dUTP). This
reaction requires a three nucleotide or longer primer with a free
3'-hydroxyl group to serve as a primer terminus for extension
(Kornberg, 1980). TdT will incorporate dNTP without the nucleotide
sequence dictated by a template. This technique allows multiple
incorporation of dig-dUTP onto the 3'-OH terminus of the s-ODN (3' end
labeling), and the s-ODN signal can be amplified. For controls, saline
or s-ODN-ddC (10 nmol) plus lipofectin in aCSF were delivered. We expected no signal derived from 3' end labeling in animals pretreated with the control s-ODN-ddC, which contained no 3'-OH terminus and,
therefore, produced no TdT-mediated signal. The incorporation of
dig-dUTP onto s-ODN by TdT and the detection of the incorporated dig-dMP on s-ODN using fluorescein conjugate of antibody against digoxigenin were performed as described in the Apop Taq kit
(Oncor, Inc., Gaithersburg, MD). The brain slices were examined under a
microscope as described previously (Liu et al., 1994).
Experimental cerebral ischemia
The effect of c-fos antisense s-ODN on the expression
of Fos, NGF, neurotrophin-3 (NT-3), and actin was examined. To
determine whether Fos expression was inhibited by the antisense
strategy described, we compared ischemic Fos expression using a well
established focal cerebral ischemia-reperfusion (FCIR) model in two
groups of animals that received c-fos antisense
(n = 22; group A) and sense (n = 16;
group S) s-ODN, respectively. Control animals received one of the
following: (1) aCSF and no FCIR (n = 17; the sham
control or group N); (2) FCIR (n = 15; the positive
control or group P); or (3) mismatch M17 s-ODN (n = 4)
in aCSF and FCIR. Briefly, 16 hr after s-ODN delivery, the animal was
anesthetized, and the right middle cerebral artery and both common
carotid arteries were occluded for 90 min. Reperfusion was initiated by
releasing the occlusion and continued for 90 min. While still under
anesthesia, the animal was killed for tissue preparations as described earlier.
The effect of s-ODN on the expression of ischemic Fos, NGF, NT-3,
and actin
The detection of ischemic Fos expression. Coronal
sections (20-µm-thick) from 20 animals were incubated in
proteinase K (10 µg/ml) for digestion at 37°C for 30 min, then in
PBS containing bovine serum albumin (BSA) (10 mg/ml) and 0.1%
NaN3 for 30 min, followed by washing in PBS-BSA five
times. The sections were then incubated with Ab-2 antibody (Oncogene
Science, Manhattan, NY) in PBS-BSA at a suitable dilution
(predetermined for its lot number) at 4°C overnight. We have found
that a 1:400 to 1:500 dilution of Ab-2 gives the best result. After
washes in PBS-BSA, the brain sections were incubated with a secondary
antibody (goat anti-rabbit IgG conjugated with biotin at 1:400
dilution) at room temperature for 45 min. The antigen-IgG complex was
incubated with avidin-horseradish peroxidase (1:100) for 30 min and
then stained with diaminobenzidine using a Vectastain ABC kit
(Vector Laboratories, Burlingame, CA). The specificity of the Fos
signal was tested using negative controls (the animal samples from
positive controls that underwent the same procedures in the absence of
the primary antibody or preadsorption of the primary antibody with Fos
antigen before immunostaining) in each determination.
Neurotrophin gene expression using ISH. We analyzed the
expression of NGF, NT-3, and -actin mRNA in the brain after FCIR in
animals previously treated with aCSF or s-ODN
(sense-rncfosr115 and anti-rncfosr115).
Because the activation of NGF mRNA is limited to neurons in the dentate
gyrus (Lindvall et al., 1992), we used ISH to determine NGF mRNA
expression in the brain tissue as described previously (An et al.,
1993), except that the cRNA transcript was labeled with
[-32P]UTP (107 cpm/ml). After
hybridization, the brain slices were digested using RNase A at 37°C
for 30 min to remove nonspecific hybrids. The specificity of the mRNA
signal was tested in animal samples from group P for their inability to
produce any signal using the [32P]mRNA (the sense)
transcript of the NGF gene.
RT-PCR. Because the background level of NGF mRNA in the
brain is ~1% of that found in the actin mRNA, we sought to amplify the hippocampal NGF and -actin genes using the RT-PCR method. We
isolated total RNA from the right (ipsilateral or ischemic) and left
(contralateral or nonischemic) hippocampus from 19 animals. The cDNA
preparation from 5 µg of total RNA was performed using 200 U of
SuperScript II (Life Technologies) as described previously (Liu, 1993). The cDNA was extracted with phenol and then with chloroform, followed by precipitation with ethanol. The cDNA was dissolved in 100 µl of TE buffer (Tris-HCl 10 mM and EDTA
1 mM, pH 8.0). One microliter of cDNA was calculated to be
equivalent to that transcribed from 1.5 ng of mRNA. The criteria of an
acceptable cDNA preparation were the expression of the
-actin gene in the cDNA sample; the -actin cDNA levels from the
RNA samples of 16 animals were not affected by FCIR, and there was no
significant difference between samples derived from the right and left
hippocampi using the hot-start PCR. To minimize the technical
feasibility of running more than 16 samples simultaneously on one
agarose gel, we pooled the cDNA from all four animals in each
treatment. Amplification of -actin in the pooled samples showed no
change in the amount of PCR product.
The primers (forward and backward primers, respectively) used for PCR
for the -actin mRNA were 5'-ccttcctgggcatggagtcctg-3' and
5'-ggagcaatgatcttgatcttc-3' (Colotta et al., 1992); for the NGF mRNA,
they were 5'-ggcaagtcagcctcttcttgcagcc-3' and
5'-gtaatgtccatgttgttctacactctg-3'; and for the NT-3 mRNA, they were
5'-gcagagcataagagtcaccg-3' and 5'-ccgatttttcttgacaaggc-3' (Maisonpierre
et al., 1991). Several technical factors can affect the amplification
result by PCR. In the preliminary studies, we found that the optimal
conditions for PCR amplification were 10 pmol of primers, 2-6
mM Mg2+ in the reaction solution, and an
annealing temperature of 55°C. In addition, we found that the PCR
product from the 3 µl cDNA sample increased linearly with increasing
numbers of cycles from 18 to 32. We used 24 cycles for the -actin
and 28 cycles for NTG amplification. For quantitative purposes, one of
the primers was 5' end-labeled with [-32P]ATP, and PCR
amplification was performed for 18 cycles. Finally, the PCR products of
the -actin, NGF, and NT-3 genes were gel-purified, and their
sequences were determined diagnostically. We found that each PCR
product contained its target sequence. Moreover, the primers
specifically amplified the target cDNA, even in the presence of other
cDNA that may share partial homology to the target gene under the PCR
conditions we used.
For PCR, the 39 µl reaction mixture containing 3 µl of cDNA and 20 pmol of primers was heated at 95°C for 5 min and then cooled to
65°C. An 11 µl mixture that contained dNTPs, buffer, and 1.5 U of
Taq polymerase was added to the reaction, which was in a thermocycler block at 65°C (Perkin-Elmer, Emeryville, CA).
Amplification was performed for no more than 30 cycles at 94°C for 45 sec, 55°C for 30 sec, and 72°C for 1 min and 30 sec. The
reaction was terminated by a 10 min extension at 70°C. Ten
microliters of each amplification reaction were resolved on a 2%
agarose gel or a 6% PAGE.
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RESULTS |
In situ uptake and distribution of s-ODNs in
rat brain
Direct detection of bio-s-ODNs using the
immunohistochemical method
To study the correlation between the distribution and the effect
of c-fos antisense s-ODN after FCIR, we first determined the
uptake and distribution of c-fos antisense s-ODN in
the right hemisphere of rat brains. After the delivery of the
bio-s-ODNs into the left lateral ventricle, we detected no significant
bio-s-ODN signal (green or yellow-green fluorescence) in the cortex
within 6 hr (n = 3) after delivery (Fig.
1A), except in a few
nuclei in the CA neurons of the hippocampus (Fig.
1D). The intensity of bio-s-ODN signals, as shown by
anti-biotin antibodies, located around the nucleus and increased with
time. The signal was noted in the cerebral cortex 2 d
(n = 2) after receiving the antisense s-ODN (Fig.
1B), whereas the signal in the hippocampal CA1 region was most intense 1 d (n = 5) after infusion (Fig.
1E). The signal in the cortex and hippocampus lasted
for at least 4 d (n = 4) (Fig.
1C,F).
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Figure 1.
The presence of bio-s-ODN in the rat brain.
Bio-s-ODN (1 nmol) with lipofectin (0.01 µg) was delivered via
intracerebroventricular route as described previously (Liu et
al., 1994). The presence of bio-s-ODN in the brain was detected by
using goat IgG against biotin and rabbit anti-goat IgG-FITC conjugates.
The nuclei were counterstained with PI and appeared red.
Representative areas in layer II of the cortex (A,
B, and C at 6, 48, and 96 hr after
delivery, respectively) and in CA1 of the hippocampus
(D, E, and F at 6, 24, and
96 hr after delivery, respectively) in the right hemisphere are shown.
The magnification in all panels is the same. Scale bar, 20 µm. The image was acquired using double exposure and Kodak
photographic film for FITC and PI signals. Because of the double
staining of FITC and PI, the biotin signal appeared
green (F) or yellow
(a blend of red and green;
B, C, E). The apparent
larger sizes of the nuclei in B-F were attributable to
the visualization of bio-s-ODNs in cell bodies in contrast to nuclear
stain only by PI in A.
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To determine whether biotin signal remained with the bio-s-ODNs in
different regions of the brain, bio-s-ODNs were recovered from the
cortex, hippocampus, and cerebellum at 1 (n = 3), 2 (n = 3), and 3 (n = 2) d after
infusion. At 3 d after delivery, we did not recover measurable
bio-s-ODN from the hippocampus or the cerebellum using dot blot
analyses. Because bio-s-ODN of 21-30 bases in length would be
recovered, the data indicate that the infused bio-s-ODN remained as
intact s-ODN for 2 d after delivery. The disappearance of brain
bio-s-ODN over time suggests degradation of the bio-s-ODN to <20 bases
in the brain.
Uptake and distribution of s-ODN using in situ labeling
of s-ODN 3'-OH terminus
Using immunohistochemical detection of bio-s-ODN, our results
indicated that some of the infused bio-s-ODNs could be observed in
nuclei of the hippocampus beginning at ~6 hr, and the intensity of
bio-s-ODN increased with time and lasted until ~4 d after infusion (Fig. 1). However, dot blot analysis demonstrated that intact s-ODNs
could not be recovered beyond 2 d after delivery. Therefore, a
direct detection of labeled s-ODN was not adequate to determine the
localization of s-ODN. To further confirm the uptake of the infused
s-ODN in the brain, we labeled the 3'-OH terminus of the s-ODN in
situ. As early as 4 hr after delivery of 1 nmol of s-ODN (n = 3), fluorescent signals were detected in the
corpus callosum and adjacent hippocampus in all three animals that
received the s-ODN (Fig.
2B), whereas no
fluorescent signal was observed in animals without surgery (the normal
control; n = 5) and in animals that received saline
(n = 2) (Fig. 2A). The intensity of
the signal indicating 3'-OH termini increased with the amount of s-ODN
delivered (10 nmol) (Fig. 2C). Larger doses of s-ODN (e.g.,
 50 nmol) led to the distribution of the signal in a wider region
beyond the lateral ventricles (data not shown). More intense and
uniform staining of ependymal hippocampal cell bodies, diffuse staining of the hippocampal neurites, and the endothelial cell bodies of the
vessel walls were observed at 1 hr after infusion in animals that
received 50 (n = 3) or 100 (n = 2) nmol
of s-ODN. The appearance of fluorescence in the neurites distinguished
the s-ODNs from the intranuclear 3'-OH labeling commonly associated
with DNA fragmentation in apoptosis. The animals that received >50
nmol of s-ODN gave excellent signals. It appeared that the infused
s-ODNs were taken up indiscriminately. However, all of the rats that
received 100 nmol showed impaired neurological function with signs of
shallow breathing and died within the first hour of s-ODN infusion. The animals that received 50 nmol of s-ODN began to show signs of shallow
breathing at 1 hr after s-ODN delivery. Therefore, we chose to use 10 nmol of s-ODN, which did not appear to have any adverse effects.
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Figure 2.
The uptake of s-ODN in the ependyma of the lateral
ventricle and beyond. The presence of s-ODNs was detected using
3'-OH-terminus labeling of TdT-mediated dig-dUTP incorporation,
followed by immunohistochemical staining using anti-dig-IgG-FITC
conjugates (Oncor, Inc.). The images were acquired using the SPOT
digital camera with a single exposure using the green (FITC) spectrum.
The anti-dig-FITC-dig-dUTP complex is shown as white
fluorescence. Four different doses of c-fos antisense
s-ODNs, each administered with 10 µg of lipofectin (1 of the 3 animals from each group is shown) in 40 µl of aCSF (A,
saline with no s-ODN; B, 1 nmol; C, 10 nmol). One of the three animals from each dosage is shown. The uptake
time was 4 hr after infusion.
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In rats that received 10 nmol of s-ODN, we examined the presence of
s-ODN at 4 hr (n = 3), 16 hr (n = 4),
1 d (n = 4), and 2 d (n = 8)
after delivery. Again, the signal in animals with 10 nmol of s-ODN at 4 hr was the same as that observed in Figure 2C and increased
with time. There was no reduction in the fluorescent signal between 16 hr (Fig. 3B) and 2 d
(Fig. 4) after infusion. The labeled
s-ODNs could be observed in the nuclei of the dentate gyrus (Fig.
3B) and the CA1 neurons (Fig. 4) in the hippocampus, the
hypothalamus, and the various layers of the cortex (Fig.
4I-VI). The s-ODNs appeared migrating from
the ependyma of the lateral ventricle into the adjacent brain
parenchyma within 1 d of delivery. Within the cells, the s-ODNs
were found in the nuclei (Fig. 3B) and as aggregates in the
perinuclear region (Fig. 4). Similar s-ODN uptake and distribution were
observed when the sense s-ODN to c-fos was delivered (10 nmol: n = 5 for 4 hr; n = 2 for 6 hr; n = 5 for 1 d; and n = 3 for
2 d). Delivery of s-ODNs without lipofectin (n = 9) showed substantially no aggregates. No signal was observed in the
control animals [saline (n = 14) or lipofectin alone
in aCSF (n = 3)], and the animals received antisense
s-ODN-ddC (10 nmol: n = 6 each at 4 hr, 1 d, and
2 d; and 50 nmol: n = 2 at 4 hr uptake times) or
in the animals that received s-ODN but without adding TdT during 3'-OH
end labeling (Fig. 3A). No 3'-OH in s-ODNs-ddC was available
for polymerization by TdT up to 2 d after infusion. These data
confirmed that the signal came from the incorporation of dig-dUTP onto
3'-OH termini of s-ODNs.
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Figure 3.
Representative digitized images of brain slices
showing the uptake of s-ODN. The presence of s-ODNs in the dentate
gyrus was detected using 3' end labeling as in Figure 2, except that 10 nmol of c-fos antisense s-ODN with 10 µg of lipofectin
in 40 µl of aCSF was delivered, and the images were acquired as in
Figure 2. The animals were killed at 16 hr after infusion. The
anti-dig-FITC-dig-dUTP complex is shown as bright white
fluorescence. Dark dots appear in the dentate gyrus of
the animals that received s-ODN but without TdT for dUTP incorporation
during the 3'-OH end labeling assay (A). Scale
bar, 20 µm.
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Figure 4.
The presence of s-ODN 48 hr after delivery. The
presence of infused s-ODNs appeared as bright white
fluorescence in one representative rat brain. Original magnifications
(40× or 400×) were as indicated. Labels indicate the
approximate cortical layers. CC, Corpus callosum. Images
were acquired as in Figure 2. Because of single staining, the nuclei
appeared as black holes.
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The effect of s-ODN on the expression of postischemic Fos, NGF,
NT-3, and actin expression
The detection of postischemic Fos expression
Very weak Fos expression was observed in the cerebral cortex and
in the dentate gyrus in all of the sham-operated controls (Fig.
5). Robust Fos expression was observed in
the cortex and hippocampus in all of animals that received saline in
the positive control (aCSF and FCIR) and in all of the animals that
received the sense s-ODN to c-fos
(sense-rncfosr115 and FCIR) (Fig. 5). Using mismatch MA17
s-ODN, we observed a slight reduction of Fos expression in the cortical
neurons (Fig. 5), indicating a loss of stringency on the MA17 s-ODN. An
inhibition of Fos expression, however, was observed in all FCIR animals
that received the antisense s-ODN to c-fos
(anti-rncfosr115). The knockdown of postischemic Fos
expression in the dentate gyrus and the cortex by the antisense s-ODN
to c-fos is in agreement with the robust uptake of the same s-ODN or bio-s-ODN in the same regions noted above (Figs.
3B, 4, respectively).
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Figure 5.
The knockdown of Fos-expression after FCIR by
c-fos antisense s-ODN. All samples shown were from the
ischemic or the right cerebral cortex or hippocampus. A low basal level
of Fos immunoreactivity was noted in the cortices without ischemia
(sham controls). Significantly increased Fos expression was present in
the cerebral cortical or hippocampal neurons in FCIR animals that
received saline (positive controls), c-fos sense s-ODN
(sense-rncfosr115), or mismatch MA17. Fos
immunoreactivity was virtually absent in FCIR animals with
c-fos antisense s-ODN
(anti-rncfosr115).
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Neurotrophin gene expression using ISH
Increased postischemic Fos expression is associated with an
increase in AP-1 binding activity (An et al., 1993). The purpose of
this study was to determine whether the expression of late effector
genes was affected. Thirty-five animals were studied in two groups of
animals that underwent one of four infusion treatments and FCIR: group
N (n = 6), group P (n = 7), group S
(n = 8), and group A (n = 14). Using
ISH, we found a significant increase in postischemic NGF expression in
the FCIR-treated right (ipsilateral) dentate gyrus in all of the
positive controls (Fig. 6, P)
and five of eight animals treated with c-fos sense s-ODN
(Fig. 6, S) compared with the left (contralateral)
hippocampus. In all of the sham-operated animals without ischemia (Fig.
6, N), no difference between the expression of NGF
mRNA in the right and left dentate gyri was noted. The increase in the
NGF expression in the ipsilateral dentate gyrus of the positive and
sense s-ODN controls was significant compared with the sham-operated
controls (Fisher's exact test; p < 0.05). In all FCIR
animals that received c-fos antisense s-ODN (Fig. 6,
A), NGF mRNA expression in the dentate gyrus
(arrow) was not statistically different from that in the
sham-operated group but was noticeably less than that observed in all
of the animals with FCIR and in five of eight that received c-fos sense s-ODN with FCIR (Fisher's exact test;
p < 0.05). The knockdown of NGF mRNA localized to the
dentate gyrus region, where there was s-ODN uptake (Fig. 3B)
and no expression of the Fos peptide (Fig. 5).
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Figure 6.
Suppression of NGF mRNA expression after FCIR by
c-fos antisense s-ODN. NGF mRNA was detected in dentate
gyri using a [32P]cRNA probe from the NGF gene.
The autoradiograms show coronal brain sections (rostral to caudal
orientation) of two representative groups of animals (sets one and two
as shown in I and II, respectively) that
underwent one of four infusion treatments and FCIR. P,
Positive controls (saline and FCIR); N, sham controls
(no FCIR); S, c-fos sense s-ODN;
A, c-fos antisense s-ODN. The
arrows in A indicate FCIR-treated right
dentate gyrus (see also Fig. 3B, s-ODN uptake; Fig. 5,
Fos knockdown). Tissue from the sense-rncfosr115 group was
hybridized with a [32P]mRNA probe; no signal was
observed (data not shown).
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Differential expression of NGF and NT-3 mRNA after FCIR
The expression of the hippocampal -actin mRNA among the four
groups of animals did not differ more than threefold (Fig.
7); therefore, the expression of the
-actin mRNA was used as the control to determine the relative
expression of hippocampal NGF mRNA. Using the coamplification RT-PCR
(Fig. 7), the amount of hippocampal NGF mRNA (lane A)
from the FCIR animals with the antisense s-ODN was no more than that in
the non-FCIR control (lane N) but was less than
that in the positive and sense controls (lanes P, S). Because there could be an interference of various
substrates that might reduce the amplification of NGF mRNA from the
animals that received c-fos antisense s-ODN during the
co-amplification, we tested single gene amplification (Fig.
8). Furthermore, the NGF mRNA was
quantitatively examined in each set of RT-PCR experiments. The amount
of NGF products from three hippocampi using RT-PCR gave a linear
response curve (r = 0.95) using different amounts of
cDNA that were transcribed from 0.75-9 ng of hippocampal mRNA. From
this standard curve, we found that a greater than fivefold increase in
the intensity of NGF products (from 200 to 900 cpm amplified from 0.75 to 3 ng, respectively) using this method was highly significant by
t test (p < 0.01). On the other
hand, a less than threefold increase in the intensity (from 900 to 2400 cpm amplified from 3 to 9 ng of mRNA, respectively) was not
statistically significant. Figure 8A shows that
postischemic NGF expression in the hippocampus significantly increased
by eightfold (t test; p < 0.01) in FCIR
animals receiving aCSF (n = 4; lane P)
or the sense s-ODN (n = 4; lane S)
compared with the sham controls without FCIR (n = 4;
lane N). The hippocampal NGF gene expression in
the animals that received the antisense s-ODN (n = 4;
lane A) was not significantly different from that
observed in the sham controls (lane N). The
result was similar to that observed using coamplification in Figure 7.
To determine that the observed NGF suppression after c-fos
antisense s-ODN infusion was not secondary to nonspecific toxicity of
the s-ODN, we also assessed the effect of this antisense s-ODN on
postischemic expression of the hippocampal NT-3 gene in the same cDNA
samples. Figure 8B shows that there was a twofold increase in the hippocampal NT-3 mRNA in the positive (lane
P) and sense (lane S) controls compared
with those in the antisense (lane A) and sham
(lane N) controls. The antisense s-ODN to
c-fos did not affect postischemic NT-3 expression. We
concluded that there was no induction of NGF mRNA in the dentate gyrus
of the animals that received c-fos antisense s-ODN after
FCIR.
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Figure 7.
Differential display of hippocampal NGF and actin
mRNA after FCIR by c-fos antisense s-ODN. The starting
cDNA in each lane is from a mixture of all right FCIR hippocampi in
each of the four groups (4 animals in each group). The PCR products
were analyzed using nondenaturing PAGE (6%). The treatment groups are
described in Figure 6. Lane M is the DNA size
marker.
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Figure 8.
Postischemic expression of hippocampal NGF and
NT-3 mRNA. NGF (A) and NT-3
(B) genes were individually amplified using 18 PCR cycles with a fixed amount of primer (10 pmol, one of which was
labeled with 32P). The PCR product was resolved in
nondenaturing 6% PAGE and was measured using the Betagen (Waltham, MA)
Betascope blot analyzer. Each lane contains a mixture of cDNA from four
animals that received the same treatment as those in Figures
5-7.
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DISCUSSION |
A consequence of cerebral uptake of c-fos antisense
s-ODN was the suppression of postischemic Fos expression after FCIR
insult. The Fos expression is similar to that experienced in stroke or traumatic head injury. In addition, NGF mRNA expression in the dentate
gyrus after FCIR was suppressed in animals treated with c-fos antisense s-ODN, whereas the expression of hippocampal
-actin and NT-3 genes was not affected. The gene products of several IEGs, including Fos, JunB, Jun, and cAMP response element-binding protein (Curran et al., 1984; Sagar et al., 1988; Sheng et al., 1990), are induced after cerebral ischemia (An et al., 1993; Sharp, 1994; Salminen et al., 1995; for review, see Akins et al., 1996). These
proteins form activation signals to regulate the expression of various
late effector genes (Sagar et al., 1988; Sharp et al., 1991; Gillardon
et al., 1996). For example, Fos and Jun, the gene products of
c-fos and c-jun, respectively, form a heterodimer via a leucine zipper (Halazonetis et al., 1988; Macgregor et al., 1990). This heterodimer binds the AP-1 DNA sequence and is presumed to
regulate the expression of >900 genes (Sharp, 1994), including those
coding for NGF (Zheng and Heinrich, 1988; Hengerer et al., 1990) and
for DNA repair enzymes (Scanlon et al., 1991). A number of methods,
including homologous recombination (Field et al., 1992) and RNA
excision by ribozymes (Funato et al., 1992), can successfully inhibit
c-fos gene expression. Antisense ODNs to c-fos
gene have been used to regulate the expression of its mRNA in cell
culture systems (Holt et al., 1986; Mercola et al., 1987; Nishikura and
Murray, 1987; Edwards et al., 1988; Kerr et al., 1988; Levi and Ozato,
1988; Levi et al., 1988; Schönthal et al., 1988; Colotta et al.,
1992) and in the rat brain (Chiasson et al., 1992, 1994; Heilig et al.,
1993; Dragunow et al., 1994; Hooper et al., 1994; Schlingensiepen et
al., 1994; Cirelli et al., 1995b; Quercia and Chang, 1996). Based on
these works, we used in vivo transfection of
c-fos antisense s-ODN to demonstrate the suppression of
FCIR-induced Fos-AP-1 activities (Liu et al., 1994).
The neurotrophic genes support the survival of specific sets of neurons
(Phillips et al., 1990; Wetmore et al., 1990; Snider, 1994). Expression
of hippocampal NGF mRNA has been shown to increase significantly after
cerebral ischemia (Lindvall et al., 1992), and we observed the same. In
a motor neuron injury model, Gu and colleagues (1997) demonstrated that
the increase in c-fos expression occurs before the induction
of NGF mRNA. The selective suppression of hippocampal NGF, but not
NT-3, mRNA after the administration of c-fos antisense s-ODN
suggests that Fos-AP-1 activities may have different effects on
various neurotrophins. The deletion of a DNA binding motif (such as
-tgagtca-) in the 5' regulatory region of a gene can be used to
suppress activation by its regulator (such as AP-1) but does not
guarantee a specific effect. In our study, the knockdown of Fos
expression by c-fos antisense s-ODN after FCIR and the
subsequent suppression of AP-1 activities (Liu et al., 1994), and NGF
mRNA expression in the right dentate gyrus correlated positively. The
specific suppression of NGF mRNA presented in this study as a direct
result of the inhibition of Fos induction may indicate an association
between Fos-AP-1 activities and NGF mRNA expression in the dentate
gyrus after FCIR. Alternatively, the suppression effect may be the
result of other effects caused by s-ODNs not mediated by
c-fos. This indirect effect could be rather specific on NGF
mRNA expression, however, in view of the fact that antisense
c-fos s-ODN has no effect on the suppression of actin and
NT-3 mRNA and that the sense and mismatch s-ODN have no effect on Fos
peptide and NGF mRNA activation. Regardless, that c-fos
antisense s-ODN suppresses NGF mRNA, directly or indirectly, suggests
that this antisense technique could be applied to in vivo
anti-NGF model in which a suppression of NGF is desirable. Moreover,
the possible involvement of AP-1 in the signal cascade of apoptosis
induced by tumor necrosis factor- may permit c-fos antisense s-ODN a therapeutic potential to suppress AP-1 and delay or
alter cell death (Roulston et al., 1998). Specific knockdown of peptide
translation using the antisense strategy offers an alternative method
for analyses of gene activation and regulation and for drug target
discovery and gene function analysis in whole animals in which gene
knockout technology is restricted.
The knockdown effect of c-fos antisense s-ODN on Fos-AP-1
activities and NGF mRNA demonstrated in our study correlated with the
time-dependent uptake of transfected c-fos antisense s-ODN in the rat brain. The regional distribution of the s-ODN appears to
follow a diffusion pattern from the periventricular wall toward the
brain parenchyma. The s-ODNs appeared to be primarily confined to the
ependymal layer of lateral ventricle 4 hr after delivery. The uptake of
s-ODN in the parenchyma does not preclude the possibility that the
actively dividing stem cells in the ependymal cell layer in the
ventricle take up the s-ODNs and migrate to cell layers away from the
ventricle (Rosario et al., 1997; Snyder et al., 1997). On the other
hand, the direct detection of bio-s-ODN using the immunohistochemical
method showed a time-dependent diffusion of biotin signal from the CSF
space into the brain parenchyma for up to 96 hr after delivery. Our
findings using this method are in agreement with those reported by
Whitesell et al. (1993). The fluorescent signal detected by this
method, however, does not distinguish between the degraded products and
those attached to the s-ODN (Fig. 1). Therefore, we used the dot blot
method to assess the content of intact bio-s-ODN after Sephadex G-25 column filtration. We observed that the cerebral bio-s-ODN uptake appeared to peak at 1 d and remained elevated up to 2 d after infusion. The decline in bio-s-ODN content in the brain beyond 2 d, especially in the hippocampus and cerebellum, suggests a probable
degradation of the s-ODNs. The uptake of s-ODN was further confirmed by
in situ 3'-OH-terminus labeling.
Our antisense s-ODN was designed to block protein translation by
hybridizing to mRNA. We demonstrated previously that c-fos antisense s-ODN is hybridized to cellular RNA between 6 and 41 hr after
delivery (Liu et al., 1994). The perinuclear localization of
c-fos antisense s-ODN supports these observations and
suggests the possible localization of c-fos mRNA. The
perinuclear localization we observed is consistent with the
observations of Agrawal et al. (1992) in their cell culture experiments
but is not consistent with the results observed by Whitesell et al.
(1993) in an animal study. In the latter study, the investigators
delivered a large quantity of s-ODNs to the CSF space by continuous
infusion for a minimum of 7 d using a mini-osmotic pump. Then, a
marked uptake of the s-ODNs by the astrocytes, with the majority
located in the nuclei, was noted (Whitesell et al., 1993). Differences
between our study and that by Whitesell et al. (1993) include the
method of infusion and the total amount of s-ODNs infused. Endocytosis may mediate the uptake of ODNs in cell culture conditions (Yakubov et
al., 1989). An in vivo pharmacokinetics study using
3H-ODN (12-mer) revealed that 3H-ODN given via
tail vein injection showed no cerebral uptake in rats (Mirabelli and
Crooke, 1993), suggesting that ODNs do not cross the blood-brain
barrier when given intravenously. In our study, the s-ODN appeared in
the corpus callosum and hippocampus before they appeared in the
cerebral cortex. The s-ODN signal was detectable in the hippocampus
beginning 4 hr after delivery. These findings suggest that cells closer
to the ventricular site of infusion accumulate the s-ODN earlier and in
greater quantities than do neurons more distal to the ventricle.
In this report, we have summarized our findings from interrelated
experiments that explore the mechanisms of s-ODN uptake in the brain,
with the focus on using antisense s-ODNs to regulate gene expression
after FCIR. These include the following: (1) brain cells are able to
take up s-ODNs; (2) it is possible to trace the distribution of
specific s-ODNs by various labeling techniques; (3) antisense s-ODNs
can affect target genes within 24 hr of delivery; and (4) the limited
presence (3 d) of s-ODNs allows a short-term control of their effects
in the CNS. The use of targeted s-ODN could facilitate a new avenue to
study gene regulation in the CNS in vivo and to promote
manipulation of genes under pathophysiological conditions in which
permanent gene displacement may not be desirable. Although additional
research is needed, the NGF-suppression model described here might be
used to study the in vivo association of hippocampal NGF
expression and neuronal regeneration (Hefti et al., 1989; Phillips et
al., 1991; D'Mello et al., 1992). In conclusion, we show several lines
of evidence that suggest that post-FCIR Fos knockdown could be used to
suppress the subsequent NGF mRNA expression.
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FOOTNOTES |
Received July 7, 1998; revised Nov. 30, 1998; accepted Dec. 3, 1998.
This work was completed during the term of Established Investigator
Award 96N40202N to P.K.L. from the American Heart Association. This
work was supported by National Institutes of Health Grants NS34810 to
P.K.L. and NS25545, NS28995, and NS32636 to C.Y.H., and by Office of
Naval Research Grant C4114503 to C.Y.H. We thank Y. Y. He, J. J. Xue, R. Speck, J. Wolff, A. Chen, and B. Chen for excellent
technical assistance, and Dr. W. J. Hamilton (Baylor College of
Medicine) for assistance in editing.
Correspondence should be addressed to Dr. Philip K. Liu, Department of
Neurosurgery, Baylor College of Medicine, 6560 Fannin Street, Suite
944, Houston, TX 77030.
| |
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Abstract
Introduction
