(Stroke. 2001;32:1028.)
© 2001 American Heart Association, Inc.
Original Contributions |
From the Departments of Neurosurgery (M.A.Y., M.M., G.H.S., D.M.K., G.K.S.), Neurology (M.A.Y., R.M.S.), and Biological Sciences (T.J.M., J.R.M., D.Y.H., R.M.S.) and Stanford Stroke Center (M.A.Y., M.M., G.H.S., T.J.M., D.M.K., J.R.M., D.Y.H., R.M.S., G.K.S.), Stanford University (Calif).
Correspondence to Midori A. Yenari, MD, Departments of Neurosurgery, Neurology, and Neurological Sciences, Stanford University School of Medicine, 1201 Welch Rd, MSLS Bldg B P304, Stanford, CA 94305-5487. E-mail yenari{at}stanford.edu
| Abstract |
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MethodsBipromoter, replication-incompetent herpes simplex virus vectors that encoded the genes for cabp and, as a reporter gene, lacZ were used. Sprague-Dawley rats received bilateral striatal injections of viral vector 12 to 15 hours before ischemia onset. With the use of an intraluminal occluding suture, animals were subjected to 1 hour of middle cerebral artery occlusion followed by 47 hours of reperfusion. Brains were harvested and stained with X-gal (to visualize ß-galactosidase, the gene product of lacZ). The number of remaining virally transfected, X-galstained neurons in both the ischemic and contralateral striata were counted and expressed as the percentage of surviving neurons in the ischemic striatum relative to the contralateral nonischemic striatum.
ResultsStriatal neuron survivorship among cabp-injected animals was 53.5±4.1% (n=10) versus 26.8±5.4% among those receiving lacZ (n=9) (mean±SEM; P<0.001).
ConclusionsWe conclude that viral vectormediated overexpression of CaBP leads to neuroprotection in this model of central nervous system injury. This is the first demonstration that CaBP overexpression protects neurons in a focal stroke model.
Key Words: calcium calcium-binding protein cerebral ischemia, focal cerebral ischemia, transient gene therapy
| Introduction |
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We have previously developed a defective herpes simplex viral (HSV) amplicon vector to overexpress CaBP, primarily in neurons. Vector-mediated overexpression of CaBP reduced intracellular calcium accumulation and improved cultured hippocampal neuron survival against conditions of hypoglycemia and excitotoxicity.10 11 At the in vivo level, we have shown that gene transfer of other potentially neuroprotective proteins, including Bcl-2,12 13 glucose transporter (glut-1),14 and Hsp72,15 protects neurons from stroke16 and that vector-mediated CaBP overexpression protects hippocampal neurons from toxicity induced by kainic acid (a glutamate agonist) and 3-acetylpyridine (an antimetabolite).17 By targeting striatal neurons with defective HSV vectors that encode the cabp gene, we overexpress CaBP to investigate whether CaBP may be neuroprotective in an experimental model of focal cerebral ischemia.
| Materials and Methods |
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Materials
Male Sprague-Dawley rats were obtained from Charles
River (Wilmington, Mass). Vero cells (African green monkey kidney
cells; ATCC CCL81) were obtained from American Type Culture Collection
(Rockville, Md). Lipofectamine was from Life Technologies
(Gaithersburg, Md). X-gal
(4-bromo-4-chloro-3-indolyl-b-D-galactopyranoside)
was from Molecular Probes (Eugene, Ore). HSV mutant d120 and cell line
E5 were provided by Dr N.A. DeLuca (University of Pittsburgh,
Pittsburgh, Pa).
Generation of HSV Vectors
The construction of amplicons p
22ßgal
4cabp
and p
22ßgal
4s and viral vectors has been described
elsewhere.10 18
Briefly, the amplicon plasmid p
22ßgal
4cabp contained the rat
cabp gene and the
Escherichia coli lacZ gene
under the control of the HSV
4 and
22 promoters,
respectively.10 The HSV oriS
and the "a" sequence
provide the necessary cis-signals for replication and packaging of the
amplicon DNA. p
22ßgal
4s, which is identical to
p
22ßgal
4cabp except that it lacks the
cabp sequence, was used to
generate control vectors. Vectors were generated by transfection of
p
22ßgal
4cabp or p
22ßgal
4s into E5 cells with the use of
lipofectamine according to the manufacturers protocol. Twenty-four
hours after transfection, the cells were superinfected with helper
virus d120 (HSV-1 strain
KOS)19 at multiplicities of
infection of 0.03 or 0.1. The cells were harvested when 100%
cytopathic effect developed. Stocks were further purified by
centrifugation at
1800g for 10 minutes, and the
supernatants were spun at
70 000g for 18 hours through a
25% sucrose cushion in PBS with the use of an SW41 rotor. The
resulting pellets were resuspended in PBS. The amplicon plasmids were
denoted with the prefix "p," and the defective vectors thus
generated were denoted with the prefix "v." The titers of helper
virus were determined on E5 cells with a standard plaque assay. The
titers of amplicon vectors were determined on Vero cells by quantifying
the number of ß-galactosidase (ß-gal, the gene product of
lacZ)expressing cells. For
v
22ßgal
4cabp (CaBP-expressing vector), titers were as follows:
1.1x107 infectious particles per
milliliter, with helper virus titer of
3.1x107 plaque-forming units per milliliter
(ratio of vector to helper virus was 1:2.8). For v
22ßgal
4s
(control vector), titers were 1.3x107
infectious particles per milliliter, with helper virus titer of
9.7x107 plaque-forming units per milliliter
(ratio of vector to helper virus was 1:7.5).
Vector Delivery
Animals were anesthetized with xylazine (5
mg/kg) and acepromazine (4 mg/kg) given
intraperitoneally, followed by 1.5% to 2%
halothane by face mask, and placed in stereotaxic frames.
Injection sites were identified over each hemisphere. Coordinates for
striatal injections (ischemia experiments) from bregma were as
follows: anteroposterior=0, mediolateral=3.5 mm with 2 injection
sites at dorsoventral=5 and 6 mm. Coordinates for hippocampal
injections were as follows: anteroposterior= -3.8 mm,
mediolateral=2.0 mm, and dorsoventral=1.8 mm. Each injection
consisted of 3 µL of vector.
Coexpression From the Bipromoter
Vectors
To verify coexpression of both CaBP and ß-gal from
the bipromoter vector, Vero cells and brain sections from the
ischemia experiments were used. Vero cells were transfected
with CaBP-expressing vector or control vector. Twelve hours later,
cultures were fixed with acetone/methanol (3:1; -20°C). Adjacent
brain sections from representative animals subjected to
focal cerebral ischemia (one injected with the CaBP-expressing
vector and the other injected with the control vector) were fixed in
3% paraformaldehyde and 20% sucrose for 24 hours.
Brain sections (30 µm) were cut on a cryostat and allowed to dry.
Sections were treated with Proteinase K (1:2 dilution, Dako) for 15
minutes, then washed in PBS.
After they were blocked in 10% normal horse serum containing 2.5% Triton-X 100, cultures and sections were incubated in primary antibodies against ß-gal (mouse monoclonal; 1:1000 dilution for Vero cells and 1:250 dilution for brain sections; Sigma) for 1 hour at room temperature. After they were washed in PBS, cultures and sections were incubated for 1 hour in fluorescein-conjugated goat anti-mouse antibody (1:150; Vector FI-2000). The cultures and sections were washed and blocked in 10% normal goat serum. The second primary antibody against CaBP (rabbit polyclonal; 1:1000 dilution for Vero cells [gift of K. Baimbridge]; 1:200 dilution for brain sections [Cappell]) was applied for 1 hour at room temperature, then washed. The second secondary antibody (Vero cells: rhodamine-conjugated horse anti-rabbit antibody; 1:100; brain sections: Texas Redconjugated goat anti-rabbit; 10 µg/mL) was applied for 1 hour, then washed. Cultures and brain sections were then viewed and photographed under an Olympus epifluorescence microscope.
Vector Specificity
Two additional animals were injected with control
vector (v
22ßgal
4s) into the striatum or hippocampus, as
described above. Brain sections were prepared and stained with X-gal (a
chromogenic substrate for ß-gal) and cresyl violet.
Adjacent sections were double immunofluorescence
labeled as described above. Antibodies against neuronal (monoclonal
antimicrotubule-associated protein 2 [MAP2]; 1:3000; Sigma)
or astrocyte (glial fibrillary acidic protein; 1:1000; Sigma) markers
were applied, followed by antibodies against ß-gal. The number of
double- and single (ß-gal only)-labeled cells were counted from the 3
brain sections centered on the injection site. Counts of cells from the
contralateral, nonischemic striata among 8
representative animals included in this study (5
CaBP-expressing vector injected, and 3 control vector injected) were
determined, and the percentage of neuronlike cells was computed from
the total number of X-galpositive cells.
Time Course of Reporter Gene Expression
To determine the time course of gene expression from
the vectors, striata were injected and brain sections were harvested 4
or 12 hours or 1, 2, or 7 days later. After they were postfixed in 3%
paraformaldehyde/20% sucrose solution for 1 to 2 days,
25-µm frozen sections in the coronal plane were taken at 100-µm
increments 0.5 mm anterior and 0.5 mm posterior to the
infusion sites. Sections were costained with X-gal to identify
vector-infected neurons expressing ß-gal, then counterstained with
cresyl violet. The number of X-galpositive cells was counted over 10
sections as described subsequently. Animals subjected to transient
focal ischemia were injected 12 to 15 hours before middle
cerebral artery (MCA) occlusion because peak expression from the
vectors coincided with this time point
(Figure 4
).
|
Transient Focal Cerebral Ischemia
Model
Rats weighing 280 to 300 g were
anesthetized by face mask with 2% halothane plus oxygen and
air supplied in a ratio of 0.2:0.8 L/min. Once surgical levels of
anesthesia were attained (assessed by absence of hind leg
withdrawal to pinch), halothane was decreased to 1% to 1.5%, and
anesthetic levels were reassessed every 15 minutes throughout the
remainder of the procedure. Either CaBP-expressing or control vectors
were directly injected bilaterally into the striata of rats. The
striatum was chosen for injection because this region
consistently shows signs of injury in this model. Animals were
allowed to recover; 12 to 15 hours later, they were
reanesthetized with halothane. Ischemia was induced
with an occluding intraluminal
suture.20 A cervical midline
incision was made, and the left carotid artery and branches were
isolated. The common carotid artery, external carotid artery, and
pterygopalatine artery were identified and ligated. An aneurysm
clip was placed on the proximal internal carotid artery, while an
arteriotomy was made on the distal common carotid artery. An uncoated
30-mm-long segment of 3-0 nylon monofilament suture with the tip
rounded by a flame was inserted into the arteriotomy. The
aneurysm clip was removed, and the suture was advanced under
direct visualization into the internal carotid artery approximately 19
to 20 mm from the bifurcation to occlude the ostium of the
MCA.
The occluding suture was kept in place for 60 minutes. At the end of the ischemic period, the suture was removed, and the surgical incisions were closed. The animal was allowed to recover, then transported to the intensive care unit at the animal facility for postoperative monitoring. Forty-eight hours later, the animal was euthanatized with a barbiturate overdose, then perfused transcardially with heparinized saline followed by 3% paraformaldehyde. Brain sections were prepared for histological analysis.
Cell Counts
Sections were fixed in 3%
paraformaldehyde/20% sucrose and reacted with X-gal to
identify vector-infected neurons expressing ß-gal and cresyl violet.
The number of transfected neurons was counted at x40 magnification by
an investigator blinded to treatment. While these vectors tend to
infect neurons, the vector can also be taken up by ependymal and
endothelial cells. Other small, nonspecific parenchymal
cells take up the vector as well. Therefore, cells were counted only if
they (1) were contained within the striatum, (2) were X-gal positive,
and (3) possessed characteristic neuronal morphology (possessed
processes, were larger in size, and cell body diameters were 15
to 25 µm). The number of surviving cells was expressed as the ratio
of positive blue neuronlike cells in the ischemic striatum
compared with the contralateral nonischemic
striatum.
Because the extent of vector-mediated infection is limited to only a few hundred striatal neurons within a 0.5-mm radius of the injection site, it is not expected that overall infarct size would be affected. On the other hand, improvement in vector-infected striatal neuron survival could be explained by smaller infarcts in one group compared with the other. To confirm that improved striatal neuron survival was not due to an imbalance between groups with regard to the severity of ischemia, the cresyl violetstained sections were visually inspected for injury. Brains were assigned a numeric score on a scale of 0 to 3, with 0 representing no damage and 3 representing severe damage with involvement of the striatum and surrounding cortex. Animals with no visible damage (score of 0) were excluded from the analysis. Furthermore, infarct sizes were computed from regions that failed to stain with cresyl violet from the centermost section of the infarct. This section reflected the anatomic level with the maximum amount of ischemic damage and was also centered on the injection site.
Statistical Analysis
Standard statistical methods were used to
analyze data. Differences between groups were determined with
Students t test.
Nonparametric tests (ie, Mann-Whitney) were used to compare
differences between CaBP-expressing and control groups with respect to
numeric infarct scores. Statistical significance was determined at the
P<0.05 level. All data are
presented as
mean±SEM.
| Results |
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Vector Specificity
Double labeling with neuronal markers and ß-gal
showed that many transfected cells were neurons
(Figure 3
). Within the hippocampus, 81±5% of all
ß-gallabeled cells were neurons, and within the striatum, 44±2%
of all ß-galpositive cells were neurons. Among nonneuronal cells,
approximately 9% were astrocytes (hippocampus), and other cells were
indeterminate. Transfected neurons could also be distinguished from
nonneuronal cells by morphology, and our criterion for cell counting
corresponded to this. Within the 8 nonischemic striata of
animals included in this study, 52±3% of all X-galpositive cells
could be identified as neurons. Therefore, the X-gal method could be
used to assess transgene expression in neurons, provided that a strict
morphological criterion was used.
|
Time Course of Vector Expression
Consistent with our prior
studies,12 15 16
gene expression from the control vector began within 4 to 6 hours after
striatal injection, and peak expression occurred at approximately 12
hours, with 53±12 X-galpositive cells per brain section. The number
of X-galpositive cells declined thereafter, with approximately 20 to
30 positive cells per brain section by 2 to 3 days. By 7 days after
injection, only a few positive cells remained
(Figure 4
).
Protection Against Focal
Ischemia
A total of 34 animals were studied. Ten were excluded
because of no infarct or because infarction was not contained within
the region of vector expression (5 receiving CaBP-expressing vector and
5 receiving control vector). The incidence of infarcts in this model is
likely due to the relatively brief duration of ischemia;
however, the relatively short occlusion time was used to ensure
translation of the transgenes by 48 hours. Another 5 were excluded
because they died before the end of the observation period (4 received
CaBP-expressing vector, and 1 received control vector). Most of these
animals died because of subarachnoid hemorrhage that
originated at the site of the occluding suture; therefore, the cause of
death was most likely due to technical considerations in the stroke
model and not the treatment itself. Among the 19 animals included in
the final analysis, infarct grades were no different between
treated and untreated groups, implying that the severity of
ischemia was similar between groups (mean infarct scores for
CaBP-expressing vector [n=10], 1.9±0.3; control vector [n=9],
2.3±0.2; P=0.46). Infarct
sizes (percentage of ipsilateral hemisphere) from the same animals were
also not significantly different (CaBP-expressing vector, 57.8±10%;
control vector, 55±5.7%;
P=0.83).
Striatal neuron survival was improved 2- to 3-fold among
animals receiving CaBP-expressing vector compared with animals
receiving control vector. The percent survivorship of striatal neurons
transfected with the CaBP-expressing vector was 53.5±4.1% of the
X-galpositive neurons in the control striatum, while among control
vectortreated animals, only 26.8±5.4% of the X-galpositive
striatal neurons remained
(P<0.001)
(Figures 5
and 6
). Absolute numbers of remaining striatal
neurons showed a significant 3-fold increase with CaBP overexpression
compared with the control group
(P<0.01). Within
nonischemic striata, the number of X-galpositive neurons was
similar between groups
(Table
).
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| Discussion |
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Earlier work examining CaBP expression after various central nervous system insults showed that CaBP immunoreactivity decreases as cells die,26 leading some to postulate that the presence of CaBP correlated with neuronal resistance to calcium-mediated injury. In addition, CaBP-containing neurons4 5 or astrocytes induced to express CaBP7 were more resistant to excitotoxic and ischemia-related injury than neurons that lacked CaBP. However, CaBP is normally found within CA1 and dentate granules of the adult rat hippocampus. Because CA1 hippocampal neurons are particularly vulnerable to ischemia and dentate granule cells are notably resistant,3 8 others believed that CaBP may have nothing to do with neuroprotection. In fact, some reports found that the presence of CaBP did not correlate with enhanced survival against ischemic and excitotoxic insults.8 9 27 Although ischemia is frequently associated with decreased protein synthesis, increases in CaBP mRNA have been detected in brains 6 to 12 hours after kainic acid administration and forebrain ischemia.28 However, endogenous CaBP levels may not be present in sufficiently high quantities to buffer toxic intracellular calcium levels. To directly determine whether CaBP serves a protective role against calcium-mediated insults, we artificially expressed it at sufficiently high levels and confirmed that it does indeed confer ischemic resistance. There are also reports of protection with CaBP overexpression in other models of neurodegeneration. Another group used retroviral vectors and found that gene transfer of cabp protected cultured neurons from neurotoxicity in a model of motor neuron disease by decreasing intracellular calcium rises.29 Conversely, they also found that decreasing CaBP expression with antisense oligonucleotides reversed this protection. Kindy et al30 showed that adenoviral vectors expressing CaBP protected hippocampal CA1 neurons against forebrain ischemia in gerbils. Our recent work also showed that at the in vivo level, CaBP overexpression protected against kainic acidinduced excitotoxicity and mitochondrial insults.17
Vector-mediated CaBP overexpression has been previously shown to alter neuronal synaptic responses, consistent with a calcium-buffering function.31 In our prior in vitro studies, the CaBP-expressing vector has also been demonstrated to reduce intracellular calcium responses.10 Consistent with the notion that calcium overload is damaging, we then found that CaBP overexpression protected neurons from various ischemia-like insults such as hypoglycemia and excitotoxin exposure, including N-methyl-D-aspartate, kainate, and glutamate.10 11 This protection could be observed even if the transfection occurred up to 30 minutes after insult. However, CaBP did not protect against cyanide toxicity, suggesting that the protective effects are not effective against mitochondrial toxins or that CaBP cannot protect against such severe insults. Interestingly, at the in vivo level, we did find hippocampal neuron protection against 3-acetylpyridine,17 a different mitochondrial toxin that damages neurons by uncoupling electron transport. This latter study suggests that the lack of protection against cyanide was more likely due to the insult severity. We now show that gene transfermediated CaBP overexpression in striatal neurons improves survival against transient focal cerebral ischemia. Given that ischemic injury is due to a variety of pathological processes, including excitotoxicity and mitochondrial disruption, our present results corroborate our previous findings.
In contrast to our findings, Klapstein et al32 found that CaBP-deficient mice were resistant to forebrain ischemia, suggesting that CaBP might play a detrimental role in ischemic injury. These animals displayed improved electrophysiological parameters and higher CA1 cell counts. In addition, there was less terminal deoxynucleotidyl transferasemediated dUTP biotin nick end labeling (TUNEL) staining in the remaining hippocampal neurons, suggesting that these mice were also more resistant to apoptotic death. Interestingly, there did not appear to be any differences in the levels of other calcium-binding proteins, such as parvalbumin, in this strain. The reasons for this discrepancy are not clear; however, there may be unforeseen alterations in other systems in lifelong CaBP-deficient animals to explain the observed neuroprotection. On the other hand, some investigators have reported that intracellular calcium buffering may be detrimental. Abdel-Hamid and Baimbridge33 found that artificial calcium buffers potentiated excitotoxicity in cultured hippocampal neurons by paradoxically increasing calcium influx through voltage-gated ion channels. The investigators proposed that calcium chelators in this setting were particularly detrimental when excitotoxin exposure was prolonged and could be due to such factors as loss of cell volume regulation or other actions of artificial buffers unrelated to cytosolic calcium levels. There may also be differences in the susceptibility of neurons to ischemic injury within the hippocampus, compared with the striatum, where we altered CaBP expression.
We show that a gene therapy approach to stroke treatment is possible; however, it is still limited by the number of neurons our vector is capable of transfecting. Because we are able to transfect only a few hundred cells, we were not able to alter overall infarct size using this approach. Other groups have applied gene transfer techniques by overexpressing a diffusible substance within the brain parenchyma34 or within ependymal cells.35 In this manner, the gene product can act remote from the transfection site to alter overall infarct size. Betz et al35 used an adenoviral vector to overexpress interleukin-1 receptor antagonist in ependymal cells 5 days before permanent MCA occlusion. They found that treated rats had smaller infarcts than those receiving a control vector. Similar findings were recently reported by Kitagawa et al,34 who overexpressed glial cellderived neurotrophic factor in the cortex using an adenoviral vector. Others have applied gene therapy to cerebral blood vessels with the hope of improving cerebral hemodynamics or blood vessel integrity.36 37 38 It should also be noted that we began gene transfer before MCA occlusion so that maximum expression from the vectors would coincide with the onset of ischemia. Given that this approach would have limited clinical relevance, future studies should determine whether cabp gene transfer could be applied after insult. Certainly, this would depend on the gene being expressed and whether its late expression might be expected to provide protection. In fact, we previously showed that gene transfer of the antiapoptotic protein Bcl-2 could protect striatal neurons when administered 90 minutes after stroke onset.13 Given that expression from these vectors begins 4 to 6 hours after injection and peaks 12 to 15 hours later, this would suggest a particularly long therapeutic window for this protein. Whether this applies to CaBP has yet to be determined for ischemia, although in our prior in vivo study, vector was delivered immediately after toxin administration.17 Finally, the issue of viral vector safety should be mentioned. While our initial studies have shown that this strain of replication-incompetent HSV does not cause cytotoxicity in rodent brains,16 39 this has not been systematically studied in humans and should be investigated before the initiation of clinical studies. In fact, our laboratories are currently investigating the possibility of ex vivo gene transfer to human brain tissue.
In the meantime, we show that intracellular calcium reduction with CaBP overexpression is a potential target for stroke treatment. Several recent clinical studies have examined various inhibitors of calcium entry; however, none of these studies were successful mainly because of the untoward psychomimetic and hemodynamic side effects. Therefore, intracellular calcium reduction may prove to be an alternate approach for stroke treatment.
| Acknowledgments |
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Received August 7, 2000; revision received October 19, 2000; accepted December 1, 2000.
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