(Stroke. 2001;32:1043.)
© 2001 American Heart Association, Inc.
Original Contributions |
Adenovirus-Mediated Gene Transfer to Ischemic Brain
Ischemic Flow Threshold for Transgene Expression
From the Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Correspondence to Hiroaki Ooboshi, MD, PhD, Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail ooboshi{at}intmed2.med.kyushu-u.ac.jp
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Abstract |
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Background and Purpose—Gene therapy may be a promising approach for treatment of brain ischemia, although protein synthesis is generally inhibited in ischemic conditions. Our goal in this study was to examine effects of brain ischemia on transgene expression of adenovirus-mediated gene transfer to ischemic brain.
Methods—Brain ischemia was produced by photochemical occlusion of the distal middle cerebral artery of spontaneously hypertensive rats (n=15). Ninety minutes after ischemia, adenoviral vectors encoding bacterial ß-galactosidase were injected into ipsilateral (nonischemic [I-n], peri-ischemic [I-p], and ischemic core [I-c] areas) and contralateral parietal (C) cortices. Cerebral blood flow before and during ischemia at each injected area was measured by laser-Doppler flowmetry. Expression of transgene was detected by histochemistry for semiquantitative scoring or by biochemical assay for quantitative analysis.
Results—Blood flow to the cortex decreased to 72±10% (mean±SEM) at I-n, 41±6% at I-p, and 23±3% at I-c after 10 minutes of ischemia. Expression of the reporter gene was consistently detected at C and I-n at each survival period. The semiquantitative score for transgene expression decreased according to severity of ischemia (C, 2.3; I-n, 2.6; I-p, 1.1; I-c, 0.3; mean values). ß-Galactosidase activity detected by chemiluminescent assay revealed that the values (mean±SEM) in the ischemic area (I-p, 15.9±9.2 mU/mg protein; I-c, 1.3±0.5) were significantly smaller than that of the nonischemic area (C, 45.4±6.9). Analysis of cerebral blood flow at I-p revealed that cerebral blood flow threshold for transgene expression was approximately 40% of the resting value.
Conclusions—Adenovirus-mediated gene transfer into the ischemic brain provided effective expression of transgene at the nonischemic and peri-ischemic areas. Gene transfer to the ischemic brain may be a promising approach for treatment of ischemic penumbra.
Key Words: adenovirus • cerebral ischemia • gene therapy • gene transfer • threshold • rats
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Introduction |
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Gene transfer is an attractive intervention for studies of basic mechanisms of biology and potentially for therapy of cerebrovascular disease.1 2 Several groups have accomplished gene transfer to blood vessels in vivo and ex vivo with cationic lipids3 4 5 or retroviral vectors.6 7 8 Efficiency of transgene expression in these methods, however, was not sufficiently high. Another promising vector for in vivo gene transfer is the replication-deficient adenovirus, which has several advantages over other gene transfer vectors.9 10 Adenoviral vectors accommodate a cDNA insert up to approximately 8 kb, can be grown to high titers, and efficiently infect both replicating and nonreplicating cells. Adenovirus-mediated gene transfer has been achieved to the brain11 12 13 14 and several blood vessels, including basilar and common carotid arteries.15 16 17 18
Several studies have shown the usefulness of gene transfer to the brain to protect against ischemic damage, although, in these studies, vectors have already been introduced in the brain.19 20 21 22 23 Because previous studies have shown that protein synthesis is inhibited in the ischemic brain,24 25 impaired machinery of gene expression may be critical for gene transfer. To rationalize gene therapy for brain ischemia, it is important to show efficacy of gene transfer even when vectors are administered after induction of brain ischemia.
In this experiment we delivered adenoviral vector after induction of brain ischemia. Brain ischemia was produced by photochemical occlusion of the distal middle cerebral artery (MCA) of spontaneously hypertensive rats (SHR), which provided a reproducible focal infarction with a simple procedure.26 Transgene expression was analyzed in relation to cerebral blood flow (CBF) to estimate the blood flow threshold of adenovirus-mediated gene transfer to the ischemic brain.
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Materials and Methods |
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Adenoviral Vectors
We used a replication-deficient recombinant adenovirus (AdCMVßGal) as a reporter virus. The virus was constructed with the use of a cDNA for bacterial ß-galactosidase, and the method was described previously.27 28 The DNA constructs comprised a full-length copy of the adenovirus genome of approximately 36 kb, from which the early region 1 gene (E1) was replaced by a cytomegalovirus (CMV) promoter and a cDNA for Escherichia coli ß-galactosidase gene with a simian virus 40 nuclear localization signal. Recombinant viruses were grown in human embryonic kidney (293) cells that complemented the E1 early viral promoters28 and were triple plaque purified to ensure that viral suspensions were free of wild-type viruses. Viral titer was determined by plaque assay on 293 cells. After purification, the virus was suspended in phosphate-buffered saline (PBS) with 3% sucrose and was kept at -80°C until used.
Experiment 1
Animals and Surgical Procedure
All animal procedures were approved by the Animal
Care and Use Review Committee of Kyushu University. Eleven male SHR,
aged 5 to 7 months and weighing 340 to 430 g, were used for
experiment 1. Rats were anesthetized with halothane (4% for
induction; 1.5% during the surgical preparation, via face mask; 0.75%
after intubation; and 0.5% for maintenance) in a mixture of
70% nitrous oxide/30% oxygen. The right femoral artery and vein were
cannulated with polyethylene tubing (PE-50). The rats were
endotracheally intubated with PE-240 tubing. Pancuronium bromide (an
initial dose of 0.3 mg followed by 0.1 mg every 30 minutes) was
intravenously injected, and the rats were mechanically
ventilated. Mean arterial blood pressure was continuously
monitored. Physiological variables were
determined before and 1 hour after distal MCA occlusion. Rectal and
head temperatures were maintained at 37°C and 36°C, respectively,
by means of a warming lamp and a heat pad.
Rats were mounted on a stereotaxic head holder in the prone position, and a 2-cm incision was made vertically midway between the right orbit and the right external auditory canal. The temporal muscle was separated and, under an operating microscope, a burr hole 3 mm in diameter was made 1 mm posterior to the anterior junction of the zygoma and squamosal bone, revealing the distal segment of MCA above the rhinal fissure. The dura was left intact.
CBF before and during ischemia at the parietal cortex was measured by laser-Doppler flowmetry. Burr holes, 1 mm in diameter, were made in the parietal cortices at 2 mm lateral and 1.5 mm posterior to the bregma in the contralateral to ischemic side (C) and at 2 mm (nonischemic area [I-n]), 3 mm (peri-ischemic area [I-p]), and 4 mm lateral (ischemic core area [I-c]) in the ipsilateral side. The resting CBF value of each area was regarded as baseline, and changes after induction of brain ischemia were expressed as percentages of the resting value.
Brain Ischemia
Brain ischemia was produced by photochemical
occlusion of the distal MCA of SHR as described
previously.26 A krypton
laser operating at 568 nm (Innova 301, Coherent Inc) was used to
irradiate the distal MCA at a power of 20 mW. The laser beam was
focused with a 30-cm focal length cylindrical lens (CKX 300, Newport
Corp) and positioned with a mirror onto the distal MCA. The
photosensitizing dye rose bengal (15 mg/mL in 0.9% saline; Wako Pure
Chemical) was administered intravenously to a body dose of
20 mg/kg over 90 seconds simultaneously with 4 minutes of
laser irradiation.
Injection of Adenoviral Vector
Ninety minutes after induction of ischemia,
the recombinant virus was injected into the parietal regions where
blood flow was measured (C, I-n, I-p, and I-c regions). A 27-gauge
needle on a Hamilton syringe was stereotaxically inserted
into both parietal cortices (2.0 mm in depth), and 5 µL of viral
suspension (3x1010 plaque-forming units per
milliliter) was injected over 10 minutes. The burr hole was then
covered with bone wax, and the scalp was sutured. After injection of
adenovirus, the rats were housed for 1 (n=4), 4 (n=4), or 7 (n=3) days.
Two hours after the distal MCA occlusion, the head wound was closed and
the catheters were removed. The rats were carefully weaned from the
respirator and returned to the home cage after regaining the ability to
breathe independently.
Histochemical Analysis of Gene
Expression
After the designated survival periods, the rats were
anesthetized with amobarbital (100 mg/kg IP) and perfused
transcardially with 2% paraformaldehyde and 0.2%
glutaraldehyde in PBS. The brain was removed and washed
thoroughly with PBS. The brain was cut into coronal sections (3 mm
thick) at the injected site and incubated in
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside
(X-Gal, Wako Pure Chemical) staining solution for 3 hours at room
temperature, rinsed in PBS, and postfixed with 10% formaldehyde.
Incubation with X-Gal was limited to 3 hours to prevent staining
endogenous ß-galactosidase, which may be seen in the
cytosol after longer (>4 hours) periods of
incubation.29 The slices of
fixed brain were analyzed for positive staining in the
macroscopic view. The fixed tissue was processed for paraffin
embedding, and sections (5 µm thick) were cut from the block with
microtomes, placed on slides, and counterstained with
hematoxylin-eosin.
Efficacy of transgene expression to the brain was assessed at 1, 4, and 7 days after injection of AdCMVßGal. The sections that contained the injection trail were examined for positive staining of ß-galactosidase (blue nuclei) by light microscopy. Expression of ß-galactosidase was analyzed semiquantitatively for expression score and scored as 0 (null), 1 (modest), 2 (moderate), or 3 (marked).
Experiment 2
Biochemical Assay for Transgene
Four SHR, aged 5 to 6 months, were quantitatively
analyzed for expression of transgene. In this experiment, CBF
measurement and injection of adenoviral vector were performed at C,
I-p, and I-c regions. Other procedures were similar to those in
experiment 1. Rats that survived for 7 days were used for biochemical
assay of transgene, as reported
previously.30 Briefly, rats
were perfused with ice-cold PBS, and the brain was removed, cut into
2-mm slices, and dissected on the dissecting plate into 2-mm cubes at
the injected sites. The brain block was minced with a scalpel blade and
lysed with 150 µL of lysis buffer containing 0.2% Triton X-100 and
100 mmol/L potassium phosphate, pH 7.8. The suspension of brain
tissue was centrifuged at
10 000g for 10 minutes, and
the supernatant was assayed for ß-galactosidase activity with the
Aurola GAL-XE assay kit (Wako Pure Chemical). Light emission was
measured with a luminometer (MiniLumat LB 9506, Berthold) and
calibrated with a standard curve generated with the use of purified
E coli ß-galactosidase
(Boehringer Mannheim). Protein concentrations were determined
with a Protein Assay CBB kit (Nacarai Tesque), and normalized
ß-galactosidase activity was expressed as milliunit ß-galactosidase
per milligram protein. Background values for chemiluminescence measured
in brain tissue from rats that were not transfected with the virus were
very low (1.07±0.11 mU/mg protein; n=6). Assay was duplicated in each
injected site, and the averaged values were
used.
Statistical Analysis
Data are presented as mean±SEM. Differences
in physiological variables and
ß-galactosidase activity among groups were analyzed with
ANOVA followed by Bonferroni’s post hoc
t test. CBF and grading scores
of transgene expression among the different regions were
analyzed by nonparametric Kruskal-Wallis test,
followed by Bonferroni’s post hoc
t test.
P<0.05 was regarded as
statistically significant.
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Results |
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Physiological parameters before ischemia in experiment 1 were as follows: mean arterial pressure, 167±6 mm Hg; plasma glucose, 5.5±0.6 mmol/L; hematocrit, 42±1%; pH 7.41±0.01; PCO2, 36.5±1.1 mm Hg; PO2, 101±5 mm Hg. Arterial pressure significantly increased after induction of ischemia (200±7 mm Hg). There were no significant differences in physiological variables among the groups for different survival periods. Blood flow to the cortex ipsilateral to the occlusion began to decrease within 10 minutes after focal ischemia (Figure 1
). The decrease in CBF was related to the distance
from ischemic core, and the reduction was greatest in I-c
(-77±3%), followed by I-p (-59±6%) and I-n (-28±10%). There
was no significant change in CBF in the C region (115±10%). CBF was
stable at each area until the end of the experiment. Changes of CBF
were not different among the groups for different survival
periods.
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Expression of the reporter gene was consistently
detected at C and I-n regions but occasionally detected at I-c
(Figure 2). Positive staining for ß-galactosidase was
detected in both neurons and nonneuronal cells. The time course of
semiquantitative analysis for transgene expression is
demonstrated in
Figure 3. Peak expression was observed at day 4 after
injection, although the differences did not reach the statistically
significant level. Therefore, the following analysis was
performed by combining scores of each survival period.
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The expression scores at each area
(Figure 4) showed that transgene expression was greatest at
I-n (2.6; mean) and C (2.3). The scores were significantly smaller in
I-p (1.1) and smallest at I-c (0.3). Analysis of expression
scores at I-p region in relation to CBF 30 minutes after
ischemia revealed that blood flow in the group with good
expression (score 3 or 2) (CBF, 45±8%,) was significantly greater
than that in the group with poor expression (score 0 or 1) (CBF,
31±3%) and that the ischemic threshold of transgene
expression was estimated as approximately 40% of the resting value
(Figure 5).
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Changes in physiological values and CBF
for quantitative analysis of transgene expression (experiment
2) were similar to those of experiment 1 (data not shown). Values of
biochemical assay for ß-galactosidase in the injection sites are
shown in
Figure 6. The amount of expressed protein was significantly
reduced in both I-p and I-c compared with that in
C.
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Discussion |
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In this experiment we demonstrated that adenovirus-mediated gene transfer 90 minutes after induction of brain ischemia provided effective expression of transgene in the ischemic brain. The intensity of expression was inversely associated with the severity of ischemia, and CBF threshold of transgene expression was estimated as 40% of the resting value.
Several studies reported that gene transfer was effective in reducing infarct size or attenuating neuronal damage.19 20 21 22 23 However, these studies were performed under the conditions that vectors for gene transfer were introduced in the brain before ischemic insult, ie, transgene was already expressed when brain ischemia was induced. Although these studies provide the first step for gene therapy, it is necessary to show efficacy of gene transfer even when vectors are administered after induction of brain ischemia.
Previous experiments reveal that protein synthesis starts to be inhibited when CBF decreases to <50% of the resting value or 30 mL/100 g per minute.24 25 Both transcriptional and translational processes are inhibited by ischemia. In the transcriptional process, disturbance of transport of mRNA from nucleus to cytosol31 or damage to cytoskeletal protein that attaches mRNA32 may be the key part of selective vulnerability to ischemia. In the translational process, energy-rich phosphates are needed for assembly of initiation complex and elongation of polypeptide chains. Recent studies suggest that the ischemia-induced decrease in activity of guanine nucleotide exchange factor leads to reduced activity of ternary complex, thereby providing vulnerability of hippocampal CA1 and striatum.33 Most of the current vectors for gene transfer, including adenovirus, Sendai virus, and liposomes, use expression machinery of host cells. Therefore, it is important to examine the time course and CBF threshold of transgene expression, although such studies are limited.34
In our experiment we revealed that expression of transgene in the brain was inversely associated with severity of brain ischemia. In the ischemic core (I-c region), expression of the reporter gene was severely inhibited. Thus, direct introduction of the gene transfer vector into the ischemic core may not be a promising approach of the gene therapy for stroke. Because we used a reporter gene alone in this study, we cannot exclude the possibility that introduction of a cytoprotective gene attenuates ischemic damage even in the ischemic core.
In the peri-ischemic (I-p) area, which is presumably the penumbral area in our ischemic model,35 a moderate degree of expression was shown compared with the nonischemic area. Our analysis in the peri-ischemic area revealed that CBF threshold for transgene expression was estimated as 40% of the resting CBF. The threshold level of transgene expression was lower than that of the general protein synthesis. Although the reason is not clear in our experiment, a strong promoter, such as the CMV promoter used in our expression cassette, may drive transgene expression more efficiently in ischemic conditions than constitutive gene.36 Therefore, our results suggest that gene transfer to the ischemic brain may be promising for treatment of the penumbral area even when the vector is administered after occurrence of brain ischemia.
In our results, gene transfer to the adjacent area in the ipsilateral cortex (I-n), where CBF was mildly reduced, showed good transgene expression. Therefore, transfer of gene that can express releasable or diffusible substances, including cytoprotective cytokines, to the adjacent area may be one of the most promising approaches of gene therapy for cerebrovascular disease. Previous results suggest that the ependyma in the ventricle or cerebral meninges may be one of the good targets for this purpose14 19 37
In conclusion, adenovirus-mediated gene transfer into the ischemic brain provided effective expression of transgene at the nonischemic and the peri-ischemic areas. CBF threshold for transgene expression in focal ischemia was estimated as approximately 40% of the resting value. Gene transfer to the ischemic penumbra may be one of the promising approaches for treatment of brain ischemia.
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Acknowledgments |
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This work was supported in part by a research grant in aid from the Ministry of Health and Welfare Comprehensive Research on Aging and Health (H11-008), Japan; by a grant from Sankyo Foundation of Life Science, Japan; and by a grant from Kimura Memorial Heart Foundation, Japan. We would like to thank the University of Iowa Gene Transfer Vector Core, especially Beverly L. Davidson and Richard D. Anderson, supported in part by the National Institutes of Health and the Roy J. Carver Foundation, for viral vector preparations.
Received August 23, 2000; revision received December 18, 2000; accepted December 27, 2000.
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