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The Journal of Neuroscience, March 15, 2001, 21(6):1876-1883
Antisense Knockdown of the Glial Glutamate Transporter GLT-1, But
Not the Neuronal Glutamate Transporter EAAC1, Exacerbates Transient
Focal Cerebral Ischemia-Induced Neuronal Damage in Rat Brain
Vemuganti L. Raghavendra
Rao1, 2,
Aclan
Dogan1,
Kathryn G.
Todd4,
Kellie K.
Bowen1,
Bum-Tae
Kim1, 5,
Jeffrey D.
Rothstein6, and
Robert J.
Dempsey1, 3
1 Department of Neurological Surgery and
2 Cardiovascular Research Center, University of
Wisconsin-Madison, Madison, Wisconsin 53792, 3 William S. Middleton Memorial Veterans Administration Hospital, Madison, Wisconsin
53792, 4 Department of Psychiatry, University of Alberta,
Edmonton, Canada T6G 2B7, 5 Department of Neurosurgery,
Soonchunhyang University Hospital, Seoul, Korea, and
6 Department of Neurology, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21287
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ABSTRACT |
Transient focal cerebral ischemia leads to extensive neuronal
damage in cerebral cortex and striatum. Normal functioning of glutamate
transporters clears the synaptically released glutamate to prevent
excitotoxic neuronal death. This study evaluated the functional
role of the glial (GLT-1) and neuronal (EAAC1) glutamate transporters
in mediating ischemic neuronal damage after transient middle cerebral
artery occlusion (MCAO). Transient MCAO in rats infused with GLT-1
antisense oligodeoxynucleotides (ODNs) led to increased infarct volume
(45 ± 8%; p < 0.05), worsened neurological status, and increased mortality rate, compared with GLT-1 sense/random ODN-infused controls. Transient MCAO in rats infused with EAAC1 antisense ODNs had no significant effect on any of these parameters. This study suggests that GLT-1, but not EAAC1, knockdown exacerbates the neuronal death and thus neurological deficit after stroke.
Key words:
antisense knockdown; EAAC1; focal cerebral ischemia; GLT-1; glutamate transporters; middle cerebral artery occlusion; neuronal damage; stroke
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INTRODUCTION |
After transient focal cerebral
ischemia, the progression of infarct evolution and neuronal damage
depends on the duration of ischemia and reperfusion (Lipton, 1999 ). The
size of the infarct increases up to 24 hr of reperfusion after a 2 hr
transient middle cerebral artery occlusion (MCAO) in rats (Zhang et
al., 1994). Immediately after transient MCAO, the extracellular
glutamate concentrations rapidly increase and return to baseline within 1 hr (Takagi et al., 1994; Uchiyama-Tsuyuki et al., 1994). This early
release of glutamate is critical to ischemic neuronal damage (Kato and
Kogure, 1999; Lipton, 1999).
Uptake into glia and neurons, mediated by a family of high-affinity
transporters (GLAST/EAAT1, GLT-1/EAAT2, EAAC1/EAAT3, EAAT4, and EAAT5),
is the only mechanism by which extracellular glutamate is inactivated
(Kanai et al., 1997). GLAST and GLT-1 are glial, whereas EAAC1 and
EAAT4 are neuronal (Rothstein et al., 1994; Chaudhry et al., 1995).
GLT-1 mediates the bulk of the glutamate uptake in forebrain (Robinson,
1999). Protein knockdown studies have shown that GLT-1 and GLAST, but
not EAAC1, are essential for maintaining the extracellular glutamate
below neurotoxic levels in the normal rat brain (Rothstein et al.,
1996).
In addition to clearing the released glutamate, subtypes of glutamate
transporters were also proposed to release intracellular glutamate
during ischemic conditions (Phillis and O'Regan, 1996). Seki et al.
(1999) showed that cell swelling-induced release and reversal of
astrocytic glutamate transport contribute to the increased extracellular glutamate in rat striatum after transient forebrain ischemia. Phillis et al. (2000) observed a 50% reduction in forebrain ischemia-evoked glutamate release by the application of
DL-threo- -benzyloxyaspartate (DL-TBOA),
which is a potent, competitive, nontransported blocker of
high-affinity, sodium-dependent glutamate transporters. Reversal of
neuronal glutamate transporters, probably localized in glutamatergic presynaptic terminals, was thought to be responsible for glutamate release from hippocampal slices subjected to in vitro
ischemia (Rossi et al., 2000). Roettger and Lipton (1996)
failed to observe any effect of dihyrokainate (DHK), a nontransportable
blocker of the reversed uptake by GLT-1, on ischemia-induced glutamate release from hippocampal slices. These studies suggest that GLT-1 reversal may not be responsible for increased extracellular glutamate concentrations under in vitro ischemic conditions.
Although dysfunctional glutamate reuptake has been proposed to promote
the neuronal death after global cerebral ischemia (Torp et al., 1995;
Rao et al., 2000) and hypoxic ischemia (Martin et al., 1997; Inage et
al., 1998), no studies have examined the functional significance of
glutamate transporter subtypes in precipitating the neuronal death
after focal cerebral ischemia. This study focused on the effect of
antisense knockdown of GLT-1 and EAAC1 on the infarct volume, neuronal
death, and neurological deficit in spontaneously hypertensive (SHR)
rats subjected to transient MCAO. Antisense knockdown of GLT-1,
but not EAAC1, exacerbated the ischemic infarct volume and neuronal
damage in cerebral cortex and striatum.
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MATERIALS AND METHODS |
Adult, male, SHR rats (250-300 gm; Charles River,
Wilmington, MA) were used in these studies. Rats were housed and cared
for in accordance with the Guide for the Care and Use of
Laboratory Animals, U.S. Department of Health and Human Services
Publication number 86-23 (revised 1986). All surgical procedures were
approved by the Research Animal Resources and Care Committee of the
University of Wisconsin-Madison.
Intracerebroventricular administration
of oligodeoxynucleotides
The antisense oligodeoxynucleotides (ODNs) were designed on the
basis of the known sequences of rat GLT-1 and EAAC1 (Kanai and Hediger,
1992; Pines et al., 1992) and successfully used previously to
specifically knock down the GLT-1 and EAAC1 proteins in rat brain
(Rothstein et al., 1996). Sense and random ODNs in which the proportion
of each nucleotide is identical to that of the antisense ODN were used
as controls. The sequences of the ODNs are 5'-ATC AAC CGA GGG TGC CAA
CAA TAT-3' (GLT-1 sense), 5'-ATA TTG TTG GCA CCC TCG GTT GAT-3' (GLT-1
antisense), 5'-AAT TGT GTT AGC CCC CTC TGT TGA-3' (GLT-1 random),
5'-GCT CGG GAT GCG ACT GGC-3' (EAAC1 sense), 5'-GCC AGT CGC ATC CCG
AGC-3' (EAAC1 antisense), and 5'-GCG GAT CCG TAC GCC CAG-3' (EAAC1
random). These ODNs were synthesized with a phosphorothioate backbone
and purified to analytical grade on HPLC by Oligos Etc. (Wilsonville,
OR). The ODNs were infused as described previously (Rothstein et al.,
1996). The lyophilized ODNs were reconstituted (2.5 mg/ml) in
artificial CSF (aCSF) containing (in mM) 119 NaCl, 3.1 KCl,
1.2 CaCl2, 1 MgSO4, 0.50 KH2PO4, 25 NaHCO3, 5 D-glucose, 2.2 urea, pH
7.4; dialyzed (Spectra/Por cellulose ester; molecular weight cutoff 2000; Thomas Scientific) overnight at 4°C; filtered (0.22 µm); and filled into osmotic minipumps. Each pump was connected to a
stainless steel cannula by peristaltic tubing and primed overnight at
37°C. The cannula was implanted into the lateral ventricle [coordinates: lateral,  1.5 mm; anterior-posterior, 0.8 mm; and dorsal-ventral, 4.8 mm; on the basis of the rat brain atlas of Paxinos and Watson (1998)] and secured to the skull with dental cement. The pump was placed in the skin fold on the neck of the rat
under halothane anesthesia. The osmotic minipumps used were Alzet model
2001 (Alza, Palo Alto, CA), which will pump at a rate of 1 µl/hr for
7 d. Thus, each rat received 60 µg of ODN per day (2.5 µg · µl 1 · hr1).
After 5 d of ODN infusion, rats were subjected to either transient MCAO (1 hr) or sham operation. After 1 d of reperfusion, rats were
neurologically evaluated and perfused transcardially with buffered
paraformaldehyde. The brains were then removed, post-fixed, and
cryoprotected. Proper functioning of the osmotic minipumps was
confirmed by weighing the filled pumps before implantation and
immediately after the rats were killed. The average total volume
of pumping was observed to be 139 ± 6 µl in 6 d (0.97 ± 0.04 µl/hr; n = 91). Correct placement of
the cannula into the lateral ventricle was confirmed by examining the
thionine-stained brain slices. The effect of antisense, sense, and
random ODN infusion on the levels of GLT-1 and EAAC1
proteins was evaluated by Western blotting as described previously (Rao
et al., 1998). In brief, tissue samples were homogenized in ice-cold 25 mM Tris-HCl buffer, pH 7.4, containing 2 mM EDTA and protease inhibitors [aprotinin, pepstatin-A, leupeptin, bestatin, 4-(2-aminoethyl) benzenesulfonyl fluoride, and
trans-epoxysuccinyl-L-leucylamido(4-guanidino)
butane]. The homogenate was centrifuged (70,000 × g;
30 min at 4°C), and the pellet (membranes) was resuspended in fresh
buffer. Proteins were electrophoresed on polyacrylamide gels,
transferred onto nitrocellulose, and probed with polyclonal
affinity-purified anti-GLT-1 and anti-EAAC1 antibodies and HRP-coupled
goat anti-rabbit IgG (Rothstein et al., 1994). The protein bands
detected by antibodies were visualized using the ECL Western blotting
kit (Amersham Pharmacia Biotech, Piscataway, NJ). Immunoblot
intensities were quantified by densitometric scanning using the NIH
Image program. A linear signal ratio for quantitative analysis was
determined by running protein concentration curves. For each sample,
blots were prepared twice. Before immunodetection, the blots were
stained with Ponceau-S to confirm the protein loading and transfer efficiency.
Transient focal cerebral ischemia
Animal preparation. Rats were anesthetized with
halothane (induction, 2%; maintenance, 1.2%) in an oxygen/nitrous
oxide (50:50) mixture. Animals were ventilated mechanically with a
rodent ventilator (model 683; Harvard Apparatus, South Natick, MA)
through an endotracheal tube (PE-240 polyethylene tubing). The left
femoral artery was cannulated for continuous monitoring of arterial
blood pressure and to obtain the measurements of pH,
PaO2, PaCO2,
hemoglobin, and blood glucose concentration (i-STAT; Sensor Devices,
Waukesha, WI). PaO2 and
PaCO2 were maintained between 100-200 and
30-40 mm Hg, respectively.
Transient middle cerebral artery occlusion. MCAO was
conducted by an intraluminal suture technique as described previously (Longa et al., 1989; Dogan et al., 1999). In brief, the left
common carotid artery (CCA), external carotid artery (ECA), and
internal carotid artery (ICA) were exposed through a ventral midline
incision. A 3-0 monofilament nylon suture with a rounded tip was
introduced into the ECA lumen and gently advanced to the ICA until
slight resistance was felt and a reduction in regional cerebral blood flow (rCBF) was seen. The rCBF dropped to 14-19% of the baseline in
40-50 sec and remained at that level throughout the occlusion period.
After 1 hr of occlusion, the suture was withdrawn to restore the
CCA-ICA-MCA blood flow [confirmed by laser Doppler flowmeter (Vasamedics, St. Paul, MN)]. In <5 min after the withdrawal of the
suture, the rCBF returned to the baseline level and remained unchanged
through 90 min of reperfusion. Body and cranial temperatures were
maintained with a heating blanket and a lamp at 37-38 and 36-37°C,
respectively, during the 1 hr of occlusion and 90 min of reperfusion.
After recovering from anesthesia, rats were returned to their cages
with ad libitum access to food and water.
rCBF measurement. Changes in rCBF were recorded as described
previously (Dogan et al., 1999). Before the MCAO was conducted, rats
were placed in the stereotaxic frame, and a craniectomy (4 mm in
diameter; 2-4 mm lateral and 1-2 mm caudal to bregma) was performed
with extreme care over the MCA territory using a trephine. The dura was
left intact. A laser Doppler flowmeter probe (model PD-434; Vasamedics)
was placed on the surface of the ipsilateral cortex (ischemic area) and
fixed to the periosteum with a 4-0 silk suture. The probe was connected
to a laser flowmeter device (Laserflo blood perfusion monitor BPM 403A;
TSI, St. Paul, MN). To confirm that antisense treatment had not changed
the rCBF during ischemia, end ischemic rCBF was measured in additional
cohorts by
4-iodo-[N-methyl-14C]antipyrine
([14C]AIP) autoradiography as described
previously (Alkayed et al., 1998; Rusa et al., 1999). For this, in a
separate set of sense- and antisense-infused rats, the laser Doppler
flowmeter probes were attached, arterial and venous femoral catheters
were inserted, and the MCA was occluded. At 60 min of occlusion,
arterial blood pressure, PO2,
PCO2, and pH were measured, and 40 µCi of
([14C]AIP (specific activity, 54 mCi/mmol; Amersham Pharmacia Biotech, Arlington Heights, IL) in 0.8 ml
of isotonic saline were infused intravenously for 45 sec.
Simultaneously, the arterial catheter was opened, and 15 free-flowing
20 µl samples were collected into heparin-coated tubes. With the
filament still in place and the laser Doppler indicating the occlusion,
the rat was decapitated, and the brain was snap-frozen by dipping in
isopentane cooled to 30°C and stored at 80°C. Each brain was
sectioned (20 µm thick) on a cryostat, and the sections from four
coronal levels (+0.5, 0.9, 2.1, and 3.9 from bregma) were
collected and exposed for 1 week to Hyperfilm max (Amersham
Pharmacia Biotech) together with 14C
standards. The blood samples were decolorized with tissue solubilizer, and the radioactivity was estimated by liquid scintillation
spectrometry. The autoradiographic images were digitized using the MCID
image analysis system (Imaging Research, St. Catherines, Ontario,
Canada), and the rates of rCBF were determined as previously described (Alkayed et al., 1998).
Histopathology. Each brain was sectioned coronally (40 µm
thick at an interval of 320 µm), stained with thionine, and scanned using the NIH Image program. The volume of the ischemic lesion was
computed by the numeric integration of data from 16 to 19 serial
sections in respect to the sectional interval. To account for the
cerebral edema and differential shrinkage resulting from tissue
processing, the injury volumes were corrected using the following
formula: corrected injury volume = contralateral hemisphere volume (ipsilateral hemisphere volume measured injury
volume) (Swanson et al., 1990).
Neurological evaluation. Neurological deficits were
evaluated on a six-point scale (Longa et al., 1989) before transient
MCAO and at 1 d of reperfusion (before the animals were killed) by an investigator blinded to the study groups. A score of 0 suggests no
neurological deficit (normal), 1 suggests mild neurological deficit
(failure to extend right forepaw fully), 2 suggests moderate neurological deficit (circling to the right), 3 suggests severe neurological deficit (falling to the right), and 4 suggests very severe
neurological deficit (the rat did not walk spontaneously and had a
depressed level of consciousness).
Statistical analysis. The ipsilateral value was compared
with the contralateral value and the sham-operated value using one-way ANOVA followed by Tukey-Kramer and Dunnett multiple comparisons post tests, respectively. The mean correlation (Pearson's)
coefficients for intrajudge and interjudge analyses of the
densitometric quantitations were in the range of 0.89-0.96.
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RESULTS |
Specific knockdown of GLT-1 and EAAC1 proteins by
antisense ODNs
Compared with aCSF infusion, GLT-1 antisense (but not sense or
random) infusion for 5 d significantly reduced GLT-1
immunoreactive protein levels in the cerebral cortex (66 ± 7%;
p < 0.05; n = 5 per group) and
striatum (60 ± 9%; p < 0.05; n = 5 per group) without affecting the levels of the cytoskeletal protein
-tubulin (Fig. 1). Hippocampus of the
GLT-1 antisense-infused rats also showed a 61 ± 11% decrease in
the GLT-1 protein levels. Previous studies showed that infusion of
GLT-1 antisense significantly reduces the GLT-1 protein levels in the
striatum and hippocampus, but not in the cerebellum and spinal cord
(Rothstein et al., 1996). Infusion of EAAC1 antisense (but not sense or
random) for 5 d specifically decreased EAAC1 protein levels in the
cerebral cortex (62 ± 9%; p < 0.05;
n = 5 per group) and striatum (67 ± 13%;
p < 0.05; n = 5 per group), compared
with aCSF-infused controls (Fig. 1).
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Figure 1.
Western blot analysis of GLT-1 and EAAC1 protein
levels in rats infused with GLT-1 and EAAC1 antisense, sense, and
random ODNs. The amount of protein loaded per lane was 2 µg in the
GLT-1 gels and 15 µg in the EAAC1 gels. Figure shows representative
samples from each group. CC, Cerebral cortex;
ST, striatum; CSF, artificial CSF;
S, sense ODN; R, random ODN; and
AS, antisense ODN. GLT-1 antisense infusion resulted in
a significant decrease in GLT-1 protein levels compared with GLT-1
sense/random-infused controls. Similarly, EAAC1 antisense infusion
significantly decreased EAAC1 protein levels compared with EAAC1
sense/random-infused controls. No significant changes were observed in
the -tubulin protein levels after the infusion of GLT-1 or EAAC1
sense/random/antisense.
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GLT-1 antisense knockdown increased the ischemic
infarct volume
In SHR rats, 1 hr transient MCAO and 24 hr reperfusion resulted in
an infarct in cerebral cortex (172 ± 27 mm3) and striatum (31 ± 5 mm3) with a total volume of 203 ± 28 mm3 (Table
1). Infusion of aCSF or GLT-1
sense/random resulted in no significant change in the transient
MCAO-induced infarct volume. Infusion of GLT-1 antisense led to a
significant increase in the cortical (43 ± 7%; p < 0.05), striatal (55 ± 9%; p < 0.05), and total (45 ± 9%; p < 0.05) infarct volume after
transient MCAO, compared with the sense/random-infused controls (Table
1). Infusion of EAAC1 sense/random or antisense had no significant
effect on the infarct volume after transient MCAO (Table 1). Figures
2 and 3
show the thionine-stained, serial coronal sections from the brains of
representative rats that underwent transient MCAO or sham operation
after infusion of GLT-1 (Fig. 2) and EAAC1 (Fig. 3) antisense and
sense. There were no statistically significant differences between the
groups infused with aCSF, GLT-1, or EAAC1 antisense, sense, or random
in any of the physiological parameters (mean arterial blood
pressure, pH, PaCO2,
PaO2, hemoglobin, blood glucose, temporalis
muscle, and rectal temperatures) (Table
2) and rCBF monitored during MCAO with a
laser Doppler (Fig. 4). The end-point
rCBF measured at 1 hr of MCAO, using
[14C]AIP autoradiography, was also not
significantly different between the GLT-1 sense- and antisense-infused
rats or between the EAAC1 sense- and antisense-infused rats (Fig.
5).
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Figure 2.
Thionine-stained serial coronal sections from the
brains of GLT-1 antisense and sense ODN-infused rats that underwent
transient MCAO (1 hr) or sham operation. This figure shows only the
GLT-1 sense-infused control, because there were no observable
differences between the GLT-1 sense- and random-infused groups.
Transient MCAO in GLT-1 antisense-infused rats resulted in
significantly bigger infarcts compared with GLT-1 sense-infused
controls.
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Figure 3.
Thionine-stained serial coronal sections from the
brains of EAAC1 antisense and sense ODN-infused rats that underwent
transient MCAO (1 hr) or sham operation. EAAC1 knockdown had no
significant effect on the infarct size.
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Table 2.
Physiological parameters in GLT-1 and EAAC1 sense, random,
and antisense ODN-infused rats undergoing transient MCAO
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Figure 4.
Effect of GLT-1 and EAAC1 antisense ODN infusion
on rCBF after transient MCAO. The rCBF was measured using a laser
Doppler flowmeter probe placed on the surface of the ipsilateral cortex
(ischemic area) through a craniectomy over the MCA territory. Changes
are expressed as percentage of the baseline. Values are mean ± SD. There were no statistically significant differences between the
groups. Infusion of GLT-1 and EAAC1 sense and random led to no
significant alterations in the rCBF after MCAO (data not shown).
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Figure 5.
End-point rCBF rates at 1 hr of MCAO as measured
by autoradiography using [14C]AIP in the GLT-1
sense (n = 5), GLT-1 antisense
(n = 5), EAAC1 sense (n = 4),
and EAAC1 antisense (n = 4)-infused rats. Flow
rates in the representative areas of cerebral cortex and striatum were
averaged across the contralateral and ipsilateral sides of the brain.
No significant differences were observed between GLT-1 sense- and
antisense-infused groups and EAAC1 sense- and antisense-infused groups.
The inset shows autoradiographs generated using the
coronal brain sections (+0.5, 0.9, 2.1, and 3.9 mm from bregma)
of [14C]AIP-administered GLT-1 sense- and
antisense-infused groups.
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GLT-1 knockdown exacerbated the ischemic neuronal damage
Microscopic analysis of the thionine-stained brain sections
showed no observable histopathological changes in the aCSF and GLT-1 or
EAAC1 sense/random-infused rats subjected to sham operation. No
observable differences were found between the rats that underwent MCAO
without any infusion and those infused with aCSF, GLT-1, or EAAC1
sense/random. GLT-1 antisense-infused/sham rats showed mild neuronal
damage in both cortex (Fig. 6,
top, second panel) and striatum (Fig. 6,
bottom, second panel), compared with the cortex (Fig. 6, top, first panel) and
striatum (Fig. 6, bottom, first panel) of
the GLT-1 sense-infused/sham control. Transient MCAO/reperfusion (1 hr/24 hr) in GLT-1 sense-infused rats resulted in a moderate neuronal
damage in the cortex, with a noticeable loss of layer V pyramidal
neurons, loosening of neuropil in layers V and VI, and increased glial
reactivity in the whole cortical infarct (Fig. 6, top,
third panel). In the striatum of the GLT-1 sense-infused/MCAO group, the medium-sized neurons showed ischemic morphology (shrunken, pink, and eosinophilic), whereas the large striatal neurons were normal (Fig. 6, bottom, third
panel). In the GLT-1 antisense-infused/MCAO group, there
was a severe neuronal loss in all six cortical layers, with a nearly
total loss of the large pyramidal neurons from layer V and evident
glial infiltration in the cortex (Fig. 6, top, fourth
panel). The striatum of the GLT-1 antisense-infused/MCAO
group also showed severe loss of the medium-sized neurons (Fig. 6,
bottom, fourth panel), and the surviving
neurons were either shrunken and eosinophilic or chromolytic and
hyperchromatic, surrounded by astrocytes and microglia.
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Figure 6.
Microscopic evaluation of the cerebral cortex
(top panels) and striatum (bottom panels)
from GLT-1 sense and antisense ODN-infused rats subjected to either
sham operation or transient MCAO. The cortical neuronal layers
(top panels) are indicated by
I-VI. Severe neuronal loss in all
cortical layers with a nearly total loss of the large pyramidal neurons
from layer V and evident glial infiltration can be seen in the brains
of GLT-1 antisense-infused/MCAO group. The striatum (bottom
panels) of the GLT-1 antisense-infused/MCAO group also showed
severe loss of the medium-sized striatal neurons
(arrowheads), whereas the large striatal neurons
(arrows) survived. The Figure shows only the
sense-infused control, because there was no observable difference
between sense- and random-infused groups.
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Transient MCAO after GLT-1 knockdown increased the neurological
deficit and mortality
Transient MCAO (1 hr) resulted in a mild to moderate
neurological deficit at 24 hr of reperfusion, with a mortality rate of ~14% during the first 24 hr of reperfusion (Table
3). Infusion of aCSF, GLT-1 sense
or random had no significant effect on the transient MCAO-induced
neurological deficit or mortality rate (Table 3), whereas transient
MCAO in the GLT-1 antisense-infused rats resulted in severe to very
severe neurological deficit with an ~33% mortality rate (Table
3). EAAC1 antisense infusion had no significant effect on the
transient MCAO-induced neurological deficit or mortality rate (Table
3). Sham rats infused with aCSF, GLT-1, or EAAC1 antisense/sense/random
showed no neurological deficit or significant mortality (Table 3).
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Table 3.
Mortality rates and neuroscores after sham operation and
transient MCAO in GLT-1 and EAAC1 sense, random, and antisense
ODN-infused rats
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DISCUSSION |
The results of this study show that GLT-1 knockdown increases the
susceptibility of rats to infarct development and neuronal death after
transient MCAO. Transient MCAO after GLT-1 knockdown also worsened the
neurological status and increased the mortality rate; however, the
increased size of the infarct may be responsible for these
factors. EAAC1 knockdown had no effect on the transient MCAO-induced neuronal death. This is the first study showing that GLT-1
dysfunction exacerbates ischemic neuronal damage.
Although there are at least five subtypes of glutamate transporters in
mammalian CNS (Kanai et al., 1997; Seal and Amara, 1999), GLT-1 is the
predominant subtype that carries the bulk of glutamate reuptake
(Robinson, 1999). Antisense knockdown of GLT-1 protein in normoxic rat
brain was shown to induce increased extracellular glutamate levels and
neuronal damage (Rothstein et al., 1996). Although GLT-1-deficient
transgenic mice showed selective neuronal damage in the hippocampal CA1
region and increased susceptibility to edema formation after
cold-induced cortical injury (Tanaka et al., 1997), GLAST-deficient
mice showed no apparent neuronal damage in any brain region but
developed more edema in cerebellum after cold-induced cerebellar injury
(Watase et al., 1998). These studies show that proper
functioning of both of the glial glutamate transporters is important
for preventing excitotoxic neuronal damage, but their role depends on
their known structural localization within the brain. GLT-1 and GLAST
were reported to be more abundant in cerebral cortex and cerebellum,
respectively (Storck et al., 1992; Torp et al., 1994). The present
antisense studies demonstrated that optimal functioning of GLT-1 in
cerebrum is essential for minimizing the neuronal damage after stroke.
Extracellular glutamate levels increase after transient MCAO (Takagi et
al., 1994; Uchiyama-Tsuyuki et al., 1994), which may lead to
excitotoxic neuronal death by overstimulating the NMDA receptors.
Previous studies showed that treatment with NMDA receptor ion channel
blockers, glycine site antagonists, competitive and noncompetitive NMDA
binding site antagonists, and antisense ODN against NMDA receptor NR1
subunit reduce the infarct volume, or the area of significant cell
necrosis, caused by permanent or transient focal ischemia (for review,
see Lipton, 1999). A slower rate of clearance of the released glutamate
may be the major consequence of GLT-1 knockdown. Increased release
(caused by ischemia) and decreased reuptake (caused by knockdown)
together may lead to an increase in the duration of glutamate action in
the synaptic cleft and may have caused more NMDA receptor stimulation
and exacerbated ischemic neuronal damage after GLT-1 knockdown.
Previous studies showed that dysfunctional glutamate transport and
cerebral energy failure synergistically promote excitotoxic neuronal
death (Massieu and Tapia, 1997; Massieu and Gomez-Raman, 1999;
Sanchez-Carbente and Massieu, 1999). Thus, cerebral energy failure
during the ischemic period may also be responsible for the exacerbated
neuronal damage after GLT-1 knockdown.
On the basis of observations that the nonspecific sodium-dependent
glutamate uptake blocker DL-TBOA reduces the forebrain ischemia-induced glutamate release by the same magnitude as DHK, Phillis et al. (2000) suggested that GLT-1 reversal may be an important
event in ischemic glutamate release. A recent preliminary study showed
that GLT-1 exists as two subspecies, viz GLT-1a (glial) and GLT-1b
(glial and neuronal), and the two forms are indistinguishable with
respect to their Km values, as well as
to the Ki values of L-trans-pyrrolidine-2,4-dicarboxylate,
DHK, and serine-O-sulfate (Chen et al., 2000). Seki et al.
(1999) showed that infusion of GLT-1-preferring inhibitor DHK
significantly attenuated the forebrain ischemia-induced glutamate
release from striatum and suggested GLT-1 reversal as a cause of
ischemia-induced glutamate release. It is unclear from this study
whether forebrain ischemia caused neuronal damage in striatum and the
inhibition of glutamate release by DHK led to neuroprotection. However,
infusion of DHK induces neuronal damage in the striatum and hippocampus
of normal rats (Massieu and Tapia, 1997). Longuemare and Swanson (1995)
showed that cultured astrocytes subjected to energy failure release
glutamate via the reversal of sodium-dependent uptake. However,
Ottersen et al. (1996) observed increased astrocytic glutamate content after ischemia. Recent studies also showed that DHK had no effect on
glutamate release and anoxic depolarization current in the hippocampal
slices subjected to in vitro ischemia (Roettger and Lipton,
1996; Rossi et al., 2000). These two studies indicate against GLT-1
reversal as a cause of ischemic glutamate release. The present
antisense studies show increased ischemic neuronal damage after GLT-1
knockdown, supporting the concept that GLT-1 reversal may not be
responsible for glutamate release during in vivo ischemia.
Because EAAC1 was reported to be absent in the glutamatergic
nerve terminals (Danbolt et al., 1998), its reversal may not be
responsible for ischemia-induced extracellular glutamate
concentrations. If EAAC1 reversal is responsible for glutamate release,
induction of ischemia after EAAC1 knockdown should be neuroprotective.
However, this study failed to observe either decrease or exacerbation
of ischemic neuronal damage after EAAC1 knockdown, suggesting against EAAC1 as a contributor of glutamate release after ischemia. Because the
antisense knockdown decreases EAAC1 protein by only ~65%, induction
of ischemia in EAAC1 gene knockout mice may clarify beyond a doubt the
role of EAAC1 in glutamate release and the neuronal damage beyond doubt.
Because glutamate transporters are dynamic proteins that could release
as well as reuptake glutamate, their function depends on their regional
and cellular localization, cerebral energy status, and the duration and
type of the neuronal insult. Furthermore, most of the studies proposing
transporter reversal responsible for glutamate release in ischemia were
conducted with in vitro ischemia paradigms (using either the
cultured cells or the hippocampal slices) that are very different from
the in vivo ischemia studies. An in vivo
study using a rat forebrain ischemia model showed that glutamate
transporter inhibitor
threo-3-hydroxy-DL-aspartate (TBHA) does not
block the ischemia-induced glutamate release (Heron et al., 1995).
However, this study had not demonstrated whether TBHA competes with
glutamate for the transporter sites from the inner side of the synaptic
membrane. Results of the present study suggest that whatever may be the
mechanism of glutamate release during ischemia, knockdown of GLT-1 (but
not EAAC1) exacerbates the transient MCAO-induced neuronal damage.
GLT-1 stimulators may be useful in preventing ischemic neuronal damage.
Although recent studies showed that the glial-derived neurotrophic
factor and pituitary adenylate cyclase-activating polypeptide
induce GLT-1 expression (Coccia et al., 1999; Figiel and Engele, 2000),
additional studies are needed to prove their usefulness in
neuropathological conditions associated with GLT-1 dysfunction. The
neuroprotective potential of the neural stem cells transfected with
GLT-1 to prevent excitotoxicity is under investigation and may prove to
be useful in the future (Poulsen et al., 2000).
| |
FOOTNOTES |
Received July 12, 2000; revised Dec. 29, 2000; accepted Jan. 2, 2001.
This work was supported by American Heart Association National Grant
9950086N, American Heart Association Wisconsin Affiliate Grant
9806376X, the University of Wisconsin (UW)-Madison Medical School
Research Committee Grant 161-9587, and the Department of Neurological
Surgery UW-Madison Start-up Grant to V.L.R.R., National Institutes of
Health Grants NS28000 and NS31220 to R.J.D., and a Korean Research
Foundation grant to B.-T.K. We thank Dr. Peter Lipton (University of
Wisconsin-Madison) for comments and suggestions.
Correspondence should be addressed to Dr. Vemuganti L. R. Rao,
Department of Neurological Surgery, University of Wisconsin-Madison, F4/309 CSC, 600 Highland Avenue, Madison, WI 53792. E-mail:
Vemugant{at}neurosurg.wisc.edu.
| |
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ABSTRACT
INTRODUCTION
